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Tumour-induced osteomalacia (TIO) is a rare paraneoplastic syndrome. The constellation of findings of unprovoked fractures, hypophosphataemia, urinary phosphate wasting and a negative genetic evaluation suggest a TIO diagnosis. Tumours leading to TIO are often small and difficult to localise using standard imaging studies. The68Ga-DOTATATE CT/positron emission tomography, a somatostatin receptor imaging modality, is the radiographical study of choice for localisation. It is highly sensitive and specific since tumours that cause oncogenic osteomalacia have been shown to express somatostatin receptors. Complete surgical resection is the treatment of choice; however, it may not always be feasible. Burosumab, a human anti-fibroblast growth factor-23 monoclonal antibody, is a therapeutic option in cases of unresectable TIO to normalise phosphorus levels and improve fracture healing. Our patient was initiated on burosumab, which led to healing of his fractures and profound symptomatic improvement of his pain. TIO is often undiagnosed for many years, leading to significant patient morbidity.

The LC3/GABARAP family of proteins is involved in nearly every stage of autophagy. Inhibition of LC3/GABARAP proteins is a promising approach to blocking autophagy, which sensitizes advanced cancers to DNA-damaging chemotherapy. Here, we report the structure-based design of stapled peptides that inhibit GABARAP with nanomolar affinities. Small changes in staple structure produced stapled peptides with very different binding modes and functional differences in LC3/GABARAP paralog selectivity, ranging from highly GABARAP-specific to broad inhibition of both subfamilies. The stapled peptides exhibited considerable cytosolic penetration and resistance to biological degradation. They also reduced autophagic flux in cultured ovarian cancer cells and sensitized ovarian cancer cells to cisplatin. These small, potent stapled peptides represent promising autophagy-modulating compounds that can be developed as novel cancer therapeutics and novel mediators of targeted protein degradation.

Upregulation of AKT3, but not AKT1 or AKT2, in AKT inhibitor-resistant MDA-MB-468 breast tumor cells.

Knockdown of AKT1 or AKT2 does not sensitizes AKTi-resistant breast tumor cells to MK2206.

Supplementary Figure from BRAFV600E-Driven Lung Adenocarcinoma Requires Copper to Sustain Autophagic Signaling and Processing

Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor–resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor–resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964–74. ©2016 AACR.

Knockdown of AKT3 sensitizes AKTi-resistant breast tumor cells to MK2206.

Table showing differentially expressed genes in T47D MK2206-resistant cells.

The transition metal copper (Cu) is an essential micronutrient required for development and proliferation, but the molecular mechanisms by which Cu contributes to these processes is not fully understood. Although traditionally studied as a static cofactor critical for the function of Cu-dependent enzymes, an expanding role for Cu is emerging to include its novel function as a dynamic mediator of signaling processes through the direct control of protein kinase activity. We now appreciate that Cu directly binds to and influences MEK1/2 and ULK1/2 kinase activity, and show here that reductions in MAPK and autophagic signaling are associated with dampened growth and survival of oncogenic BRAF-driven lung adenocarcinoma cells upon loss of Ctr1. Efficient autophagy, clonogenic survival, and tumorigenesis of BRAF-mutant cells required ULK1 Cu-binding. Although treatment with canonical MAPK inhibitors resulted in the upregulation of protective autophagy, mechanistically, the Cu chelator tetrathiomolybdate (TTM) was sufficient to target both autophagic and MAPK signaling as a means to blunt BRAF-driven tumorigenic properties. These findings support leveraging Cu chelation with TTM as an alternative therapeutic strategy to impair autophagy and MAPK signaling. As traditional MAPK monotherapies initiate autophagy signaling and promote cancer cell survival. Implications: We establish that copper chelation therapy inhibits both autophagy and MAPK signaling in BRAFV600E-driven lung adenocarcinoma, thus overcoming the upregulation of protective autophagy elicited by canonical MAPK pathway inhibitors.

Purpose: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies.Experimental Design: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low Km for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context.Results: LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model.Conclusions: We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies. Clin Cancer Res; 20(7); 1834–45. ©2014 AACR.

PDF file - 346KB, Supplementary Figure 1. Normal vs. Tumor expression of PIM kinases mRNA across 17 tissue types. Supplementary Figure 2. Comparison of LGB321 on-target activity vs. Previously described pan-PIM inhibitor. Supplementary Figure 3. KINOMESCANTM of 1 mu M LGB321. Supplementary Figure 4. Biochemical and cellular selectivity of the LGB321 PIM inhibitor series towards GSK3 beta. Supplementary Figure 5. LGB321 does not inhibit EGFR signaling in HCC1954 cells. Supplementary Table 1. Sensitivity to LGB321 and Erlotinib of Lung Cells in the CCLE. Supplementary Table 2. Sensitivity to LGB321 of hematological malignacies cell lines.

Supplementary figure 2. Aspirin treatment results in activation of AMPK and decrease in mTORC1 signaling.

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiologic studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA. Here, we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on COX-2 and NF-κB. We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mTORC1 inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, whereas combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports the evaluation of aspirin and PI3K pathway inhibitors as a combination therapy for targeting breast cancer. Cancer Res; 77(3); 790–801. ©2016 AACR.

Supplementary figure 1. Expression of mutant PIK3CA increases COX-2 expression and IKKbeta/NF-KB signaling.

Targeting aberrant kinase activity in cancer relies on unmasking cellular inputs such as growth factors, nutrients, and metabolites that contribute to cancer initiation and progression1. While the transition metal copper (Cu) is an essential nutrient that is traditionally viewed as a static cofactor within enzyme active sites2, a newfound role for Cu as a modulator of kinase signaling is emerging3, 4. We discovered that Cu is required for the activity of the autophagic kinases ULK1/2 through a direct Cu-ULK1/2 interaction. Genetic loss of the Cu transporterCtr1or mutations in ULK1 that disrupt Cu-binding reduced ULK1/2-dependent signaling and autophagosome complex formation. Elevated intracellular Cu levels are associated with starvation induced autophagy and sufficient to enhance ULK1 kinase activity and in turn autophagic flux. Targeting autophagy machinery is a promising therapeutic strategy in cancers5, but is limited by the absence of potent inhibitors and the emergence of resistance. The growth and survival of lung tumors driven by KRASG12Dis diminished in the absence ofCtr1, depends on ULK1 Cu-binding, and is associated with reduced autophagy levels and signaling. These findings suggest a new molecular basis for exploiting Cu-chelation therapy to forestall autophagy signaling to limit proliferation and survival in cancer.

The principal unmet need in BRAFV600E-positive melanoma is lack of an adequate therapeutic strategy capable of overcoming resistance to clinically approved targeted therapies against oncogenic BRAF and/or the downstream MEK1/2 kinases. We previously discovered that copper (Cu) is required for MEK1 and MEK2 activity through a direct Cu–MEK1/2 interaction. Repurposing the clinical Cu chelator tetrathiomolybdate (TTM) is supported by efficacy in BRAFV600E-driven melanoma models, due in part to inhibition of MEK1/2 kinase activity. However, the antineoplastic activity of Cu chelators is cytostatic. Here, we performed high-throughput small-molecule screens to identify bioactive compounds that synergize with TTM in BRAFV600E-driven melanoma cells. Genetic perturbation or pharmacologic inhibition of specific members of the BCL2 family of antiapoptotic proteins (BCL-W, BCL-XL, and MCL1) selectively reduced cell viability when combined with a Cu chelator and induced CASPASE-dependent cell death. Further, in BRAFV600E-positive melanoma cells evolved to be resistant to BRAF and/or MEK1/2 inhibitors, combined treatment with TTM and the clinically evaluated BCL2 inhibitor, ABT-263, restored tumor growth suppression and induced apoptosis. These findings further support Cu chelation as a therapeutic strategy to target oncogene-dependent tumor cell growth and survival by enhancing Cu chelator efficacy with chemical inducers of apoptosis, especially in the context of refractory or relapsed BRAFV600E-driven melanoma. Significance: This study unveils a novel collateral drug sensitivity elicited by combining copper chelators and BH3 mimetics for treatment of BRAFV600E mutation-positive melanoma.

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth., eLife digest Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.

The PI3K/Akt pathway is frequently deregulated in human cancers, and multiple Akt inhibitors are currently under clinical evaluation. Based on the experience from other molecular targeted therapies, however, it is likely that acquired resistance will be developed in patients treated with Akt inhibitors. We established breast cancer models of acquired resistance by prolonged treatment of cells with allosteric or ATP-competitive Akt inhibitors. Phospho-Receptor tyrosine kinase (Phospho-RTK) arrays revealed hyper-phosphorylation of multiple RTKS, including EGFR, Her2, HFGR, EhpB3 and ROR1, in Akt-inhibitor-resistant cells. Importantly, resistance can be overcome by treatment with an EGFR inhibitor. We further showed that cancer stem cells (CSCs) are enriched in breast tumor cells that have developed resistance to Akt inhibitors. Several candidates of CSC regulators, such as ID4, are identified by RNA sequencing. Cosmic analysis indicated that sensitivity of tumor cells to Akt inhibitors can be predicted by ID4 and stem cell/epithelial–mesenchymal transition pathway targets. These findings indicate the potential of targeting the EGFR pathway and CSC program to circumvent Akt inhibitor resistance in breast cancer.

Copper is essential for life, and beyond its well-established ability to serve as a tightly-bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low molecular weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a tris[(2-pyridyl)methyl]amine (TPA) bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and GSH/GSSG ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader crosstalk between metal and redox pathways in health and disease with activity-based probes.SignificanceCopper is a required metal nutrient for life, yet its altered homeostasis is associated with many diseases. Thus, to develop new methods to help decipher copper biology, we present an activity-based ratiometric FRET probe that exploits a biomimetic, copper(I)-dependent cleavage reaction to enable imaging of loosely-bound, labile copper pools in cells with metal and oxidation state selectivity and a self-calibrating ratiometric response. Application of this technology to cellular models of cancer reveals that oncogene-driven changes in the metabolism of glutathione, a major cellular redox buffer, leads to a labile copper(I) deficiency. This work establishes the relevance of copper dysregulation to cancer metabolism and presages further opportunities for activity-based sensing in studies of metal biology.

Copper (Cu) is a tightly regulated micronutrient that functions as a structural or catalytic cofactor for specific proteins essential for a diverse array of biological processes. While the study of the extremely rare genetic diseases, Menkes and Wilson, has highlighted the requirement for proper Cu acquisition and elimination in biological systems for cellular growth and proliferation, the importance of dedicated Cu transport systems, like the Cu chaperones ATOX1 and CCS, in the pathophysiology of cancer is not well defined. We found that ATOX1 was significantly overexpressed in human blood, breast, and skin cancer samples, while CCS was significantly altered in human brain, liver, ovarian, and prostate cancer when compared to normal tissue. Further analysis of genetic expression data in Cancer Cell Line Encyclopedia (CCLE) revealed that ATOX1 is highly expressed in melanoma cell lines over other cancer cell lines. We previously found that Cu is required for BRAFV600E-driven MAPK signaling and melanomagenesis. Here we show that genetic loss of ATOX1 decreased BRAFV600E-dependent growth and signaling in human melanoma cell lines. Pharmacological inhibition of ATOX1 with a small molecule, DCAC50, decreased the phosphorylation of ERK1/2 and reduced the growth of BRAF mutation-positive melanoma cell lines in a dose-dependent manner. Taken together, these results suggest that targeting the Cu chaperone ATOX1 as a novel therapeutic angle in BRAFV600E-driven melanomas.

Metals are vital for life as they are necessary for essential biological processes. Traditionally, metals are categorized as either dynamic signals or static cofactors. Redox-inactive metals such as calcium (Ca), potassium (K), sodium (Na), and zinc (Zn) signal through large fluctuations in their metal-ion pools. In contrast, redox-active transition metals such as copper (Cu) and iron (Fe) drive catalysis and are largely characterized as static cofactors that must be buried and protected within the active sites of proteins, due to their ability to generate damaging reactive-oxygen species through Fenton chemistry. Cu has largely been studied as a static cofactor in fundamental processes from cellular respiration to pigmentation, working through cytochrome c oxidase and tyrosinase, respectively. However, within the last decade, a new paradigm in nutrient sensing and protein regulation - termed 'metalloallostery' - has emerged, expanding the repertoire of Cu beyond the catalytic proteins to dynamic signaling molecules essential for cellular processes that impact normal physiology and disease states. In this Primer we introduce both the 'traditional' and emerging roles for Cu in biology and the many ways in which Cu intersects with human health.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

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SummaryNormal physiology relies on the precise coordination of intracellular signal transduction pathways that respond to nutrient availability to balance cell growth and cell death. We recently established a critical mechanistic function for the redox-active micronutrient copper (Cu) in the canonical mitogen activated protein kinase (MAPK) pathway at the level of MEK1 and MEK2. Here we report the X-ray crystal structure of Cu-MEK1 and reveal active site chemical ligands and oxidation state specificity for MEK1 Cu coordination. Mechanistically, the Cu chaperone CCS selectively bound to and facilitated Cu transfer to MEK1. Mutations in MEK1 that disrupt Cu(I) affinity or a CCS small molecule inhibitor reduced Cu-stimulated MEK1 kinase activity. These atomic and molecular level data provide the first mechanistic insights of Cu kinase signaling and could be exploited for the development of novel MEK1/2 inhibitors that either target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

Background Adverse drug events (ADEs) often occur as a result of drug-drug interactions (DDIs). The use of data mining for detecting effects of drug combinations on ADE has attracted growing attention and interest, however, most studies focused on analyzing pairwise DDIs. Recent efforts have been made to explore the directional relationships among high-dimensional drug combinations and have shown effectiveness on prediction of ADE risk. However, the existing approaches become inefficient from both computational and illustrative perspectives when considering more than three drugs. Methods We proposed an efficient approach to estimate the directional effects of high-order DDIs through frequent itemset mining, and further developed a novel visualization method to organize and present the high-order directional DDI effects involving more than three drugs in an interactive, concise and comprehensive manner. We demonstrated its performance by mining the directional DDIs associated with myopathy using a publicly available FAERS dataset. Results Directional effects of DDIs involving up to seven drugs were reported. Our analysis confirmed previously reported myopathy associated DDIs including interactions between fusidic acid with simvastatin and atorvastatin. Furthermore, we uncovered a number of novel DDIs leading to increased risk for myopathy, such as the co-administration of zoledronate with different types of drugs including antibiotics (ciprofloxacin, levofloxacin) and analgesics (acetaminophen, fentanyl, gabapentin, oxycodone). Finally, we visualized directional DDI findings via the proposed tool, which allows one to interactively select any drug combination as the baseline and zoom in/out to obtain both detailed and overall picture of interested drugs. Conclusions We developed a more efficient data mining strategy to identify high-order directional DDIs, and designed a scalable tool to visualize high-order DDI findings. The proposed method and tool have the potential to contribute to the drug interaction research and ultimately impact patient health care. Availability and implementation http://lishenlab.com/d3i/explorer.html

Introduction: Hypophosphatasia (HPP) is a rare inherited disease of mineral metabolism characterized by low activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNALP), which causes an inability to liberate inorganic phosphate for hydroxyapatite crystal propagation as well as toxic accumulation of inorganic pyrophosphate, pyridoxal-5’-phosphate and urinary phosphoethanolamine. It has a prevalence of 1/100,000 to 1/900,000, although milder forms have an estimated prevalence of 1/6,370. Variable mutations in TNALP cause clinical expressions ranging from a severe perinatal form, which is often fatal after birth from pulmonary complications, to an infantile form, which can cause vitamin B6-responsive seizures, to an asymptomatic adult form. Case: A 27-year-old, ventilator-dependent female with osteoporosis, hypothyroidism, cerebral palsy with previous spinal fusion, seizure disorder and nephrocalcinosis presented with surgical site infection from a right femur ORIF she underwent a month ago. She had a history of microfractures and low-impact fractures of both femurs requiring several surgeries. Osteoporosis was diagnosed at age 5 and she had been on Fosamax ever since. She did not meet any developmental milestones as a baby and never reached menarche. Various diagnoses by multiple specialists did not fully explain her clinical presentation. Her medications included alendronate 5 mg, calcium carbonate 600 mg, ergocalciferol 400 U and levothyroxine 50 mcg. Physical exam showed poor dentition, a misshapen skull and bowed legs with contractures of her extremities. Her labs revealed Ca 9.1 mg/dL (8.4–10.2 mg/dL), albumin 3.2 gm/dL (3.5–5.2 gm/dL), phosphorus 5.1 mg/dL (2.5–4.5 mg/dL), alkaline phosphatase 32 U/L (35–105 U/L), PTH 28 pg/mL (15–65 pg/mL), vitamin D 33.5 ng/mL (30–100 ng/mL), C-telopeptide 509 pg/mL (34–635 pg/mL). A right knee X-Ray reported diffusely gracile and demineralized bones with muscular atrophy. She recently transitioned care from a pediatric endocrinologist to an adult endocrinologist, who tested her positive for heterozygous ALPL pathogenic variant hypophosphatasia and was considering her for asfotase alfa enzyme replacement therapy. Discussion: Our patient had infantile HPP, but due to misdiagnosis as osteoporosis, she was inappropriately treated with a bisphosphate for over 20 years. Treatment of HPP had been supportive until the approval of asfotase alfa (Strensiq) in October 2015. It is a bone-targeted human recombinant enzyme replacement therapy approved for infantile- and juvenile-onset HPP and has been shown to decrease mortality from 73% to 16% at age 5. With improvement in life-sustaining technology, more HPP patients are able to survive into adulthood. Awareness of the complex and polymorphic presentation of HPP by adult endocrinologists is paramount for accurate diagnosis, thus avoiding inappropriate treatments.

Summary: A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients. : Hyrina et al. employ a non-biased functional CRISPR screening approach to identify host factors regulating HBsAg expression as well as those targeted by RG7834, a HBsAg inhibitor. The screen highlighted over 60 genes and identified a mechanism by which ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing. Keywords: HBV, HBsAg, CRISPR, genome-wide screen, RG7834, ZCCHC14, TENT4B, RNA tailing

Acquired resistance to molecular targeted therapy represents a major challenge for the effective treatment of cancer. Hyperactivation of the PI3K/AKT pathway is frequently observed in virtually all human malignancies, and numerous PI3K and AKT inhibitors are currently under clinical evaluation. However, mechanisms of acquired resistance to AKT inhibitors have yet to be described. Here, we use a breast cancer preclinical model to identify resistance mechanisms to a small molecule allosteric AKT inhibitor, MK2206. Using a step-wise and chronic high-dose exposure, breast cancer cell lines harboring oncogenic PI3K resistant to MK2206 were established. Using this model, we reveal that AKT3 expression is markedly upregulated in AKT inhibitor–resistant cells. Induction of AKT3 is regulated epigenetically by the bromodomain and extra terminal domain proteins. Importantly, knockdown of AKT3, but not AKT1 or AKT2, in resistant cells restores sensitivity to MK2206. AKT inhibitor–resistant cells also display an epithelial to mesenchymal transition phenotype as assessed by alterations in the levels of E-Cadherin, N-Cadherin, and vimentin, as well as enhanced invasiveness of tumor spheroids. Notably, the invasive morphology of resistant spheroids is diminished upon AKT3 depletion. We also show that resistance to MK2206 is reversible because upon drug removal resistant cells regain sensitivity to AKT inhibition, accompanied by reexpression of epithelial markers and reduction of AKT3 expression, implying that epigenetic reprogramming contributes to acquisition of resistance. These findings provide a rationale for developing therapeutics targeting AKT3 to circumvent acquired resistance in breast cancer. Mol Cancer Ther; 15(8); 1964–74. ©2016 AACR.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency. We characterized the structural basis for inhibition using NMR methods and showed that the compounds stabilized the inactive conformation of KRASG12V. Determination of the biophysical affinity of binding using biolayer interferometry demonstrated that the potency of inhibition matches the affinity of binding only when KRAS is in its native state, namely post-translationally modified and in a lipid environment. The assays we describe here provide a first-time alignment across biochemical, biophysical, and cellular KRAS assays through incorporation of key physiological factors regulating RAS biology, namely a negatively charged lipid environment and prenylation, into the in vitro assays. These assays and the ligands we discovered are valuable tools for further study of KRAS inhibition and drug discovery., PLoS ONE, 12 (4), ISSN:1932-6203

Pancreatic ductal adenocarcinoma (PDA) remains one of the most difficult cancers to effectively treat. Over 90% of PDA are driven by oncogenic KRAS, which leads to metabolic rewiring in the cell, including increased glucose uptake and increased flux through the hexosamine biosynthesis pathway (HBP). The HBP, which branches off from glycolysis at fructose-6-phosphate and also requires glutamine, acetyl-CoA, and uridine triphosphate, is responsible for synthesizing uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. UDP-GlcNAc can also be generated through recycling of GlcNAc via N-acetylglucosamine kinase (NAGK). Glycans are important for maintaining protein homeostasis and cell signaling. However, while there is increased HBP flux in PDA and glycosylation is elevated in a range of cancers, how this is regulated and the functional significance of increased glycosylation in cancer are not well understood. Because of the multiple metabolic inputs required for UDP-GlcNAc synthesis, the HBP has been proposed as a nutrient sensing pathway. Surprisingly, however, we found that PDA cells robustly maintain both O-GlcNAc and N-glycosylation when exposed to nutrient deprivation. The enzyme N-acetylglucosamine kinase (NAGK) is upregulated under low glutamine conditions, and through stable isotope tracing studies, we demonstrate that PDA cells are able to switch to use of the salvage pathway over the HBP for generation and maintenance of the UDP-GlcNAc pool via NAGK. Together, this work points to a mechanism of metabolic flexibility in generating UDP-GlcNAC that allows PDA cells to grow in a nutrient-poor environment. Citation Format: Sydney L. Campbell, Clementina A. Mesaros, Tiffany Tsang, Michael Noji, Salisa Kruijning, Sophie Trefely, Luke Izzo, Ian A. Blair, Kathryn E. Wellen. Glutamine deprivation triggers hexosamine salvage in pancreatic cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C10.

The Children’s Measurement Framework (CMF) is a new indicator-based tool for monitoring children’s equality and human rights in Britain. The article provides a conceptual and methodological overview of the CMF and presents initial findings against a subset of indicators. We begin by locating the CMF within the growing body of research that uses statistical indicators to monitor child well-being; and by considering the Framework’s conceptual and methodological positioning in the light of the broader themes discussed in the broader literature on child indicators. The theoretical underpinnings of the CMF in Sen's capability approach are next explored. The CMF indicator set, comprising 50 statistical indicators for monitoring children's equality and human rights across 10 critical domains, is described. Finally, illustrations of CMF indicators are provided for the life, health, education and physical security domains drawing on a range of administrative and social survey data sources.

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiological studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA. Here we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on cyclooxygenase-2 (COX-2) and nuclear factor-kappa B (NF-κB). We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mammalian target of rapamycin complex 1 (mTORC1) inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, while combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports evaluation of aspirin and PI3K pathway inhibitors as combination therapy for targeting breast cancer.

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor–enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 °C. van’t Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.

Purpose: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies. Experimental Design: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low Km for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context. Results: LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model. Conclusions: We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies. Clin Cancer Res; 20(7); 1834–45. ©2014 AACR.