Epinephrine inhibits PI3Kα via the Hippo kinases.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

Obesity promotes breast epithelium DNA damage in women carrying a germline mutation in BRCA1 or BRCA2.

Obesity, defined as a body mass index (BMI) ≥ 30, is an established risk factor for breast cancer among women in the general population after menopause. Whether elevated BMI is a risk factor for women with a germline mutation in BRCA1 or BRCA2 is less clear because of inconsistent findings from epidemiological studies and a lack of mechanistic studies in this population. Here, we show that DNA damage in normal breast epithelia of women carrying a BRCA mutation is positively correlated with BMI and with biomarkers of metabolic dysfunction. In addition, RNA sequencing showed obesity-associated alterations to the breast adipose microenvironment of BRCA mutation carriers, including activation of estrogen biosynthesis, which affected neighboring breast epithelial cells. In breast tissue explants cultured from women carrying a BRCA mutation, we found that blockade of estrogen biosynthesis or estrogen receptor activity decreased DNA damage. Additional obesity-associated factors, including leptin and insulin, increased DNA damage in human BRCA heterozygous epithelial cells, and inhibiting the signaling of these factors with a leptin-neutralizing antibody or PI3K inhibitor, respectively, decreased DNA damage. Furthermore, we show that increased adiposity was associated with mammary gland DNA damage and increased penetrance of mammary tumors in Brca1+/- mice. Overall, our results provide mechanistic evidence in support of a link between elevated BMI and breast cancer development in BRCA mutation carriers. This suggests that maintaining a lower body weight or pharmacologically targeting estrogen or metabolic dysfunction may reduce the risk of breast cancer in this population.

An atlas of substrate specificities for the human serine/threonine kinome.

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

PI3K drives the de novo synthesis of coenzyme A from vitamin B5.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K-PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

PI3King the Environment for Growth: PI3K Activation Drives Transcriptome Changes That Support Oncogenic Growth.

PI3K signaling plays an integral role in cells, coordinating the necessary alterations in cellular metabolism and programs to support survival and proliferation. In the first genome-wide analysis of alternative splicing in PIK3CA-mutant breast cancer, Ladewig and colleagues show that activating mutations in PIK3CA alter the use of known exons and splice junctions, leading to changes in gene expression and transcription factor activity that promote an oncogenic phenotype. Their work reveals a novel mechanism underlying the functional impact of PI3K signal activation in the context of breast cancer, where PIK3CA mutations are common and PI3K inhibitors are part of the standard of care. Their studies uncover a feedforward mechanism by which PI3K signaling enables a shift in the spectrum of translated splice variants as another method through which the PI3K pathway has evolved to regulate its own activity, thereby modifying the cellular response to upstream activation based on the signaling that has come before. These findings have profound implications for understanding the evolution of the PI3K pathway and the rewiring of cells in response to prolonged or repeated signal activation. See related article by Ladewig et al., p. 2269.

Occipital Headache Evaluation and Rates of Migraine Assessment, Diagnosis, and Treatment in Patients Receiving Greater Occipital Nerve Blocks in an Academic Pain Clinic.

OBJECTIVE: Diagnosis of patients with occipital headache can be challenging, as both primary and secondary causes must be considered. Our study assessed how often migraine is screened for, diagnosed, and treated in patients receiving greater occipital nerve blocks (GONBs) in a pain clinic. DESIGN: Institutional review board-approved, retrospective observational study. SETTING: Academic multidisciplinary pain clinic. SUBJECTS: One hundred forty-three consecutive patients who received GONBs. RESULTS: About 75% of patients had been evaluated by neurologists and about 25% by non-neurologist pain specialists only, and 62.2% of patients had photophobia, phonophobia, and nausea assessed. Compared with patients who had been evaluated by non-neurologists, patients who had been evaluated by a neurologist were more likely to have photophobia, phonophobia, and nausea assessed (75.9% vs 20.0%, odds ratio [OR] 12.6, 95% confidence interval [CI] 4.90 to 32.2); more likely to be diagnosed with migraine (48.1% vs 14.3%, OR 5.6, 95% CI 2.0 to 15); less likely to be diagnosed with occipital neuralgia (39.8% vs 65.7%, OR 0.3, 95% CI 0.2 to 0.8); and equally likely to be diagnosed with cervicogenic headache (21.3% vs 25.7%, OR 0.8, 95% CI 0.3 to 1.9). Among patients diagnosed with migraine, 82.5% received acute migraine treatment, 89.5% received preventive migraine treatment, and 52.6% were documented as receiving migraine lifestyle counseling. CONCLUSIONS: Of the patients in this study who had occipital headache and received GONBs, 62.2% were assessed for migraine, and most received appropriate acute, preventive, and lifestyle treatments when diagnosed. Patients seen by neurologists were significantly more likely to be screened for and diagnosed with migraine than were those evaluated by non-neurologist pain medicine specialists only. All clinicians should remain vigilant for migraine in patients with occipital headache.

Circling back to PTEN: Fumarate inhibits canonical tumor suppressor.

Ge et al. (2022) describes an inhibitory, post-translational modification of PTEN at C211 by fumarate, which offers new insight into the integration of PI3K signaling and metabolism via a potential feedforward regulatory mechanism involving a PI3K-glucose-fumarate-PTEN axis.

Patient-Derived In Vitro and In Vivo Models of Cancer.

Over the last two decades, cancer researchers have taken the promise offered by the Human Genome Project and have expanded its capacity to use sequencing to identify the genomic alterations that give rise to and sustain individual tumors. This expansion has allowed researchers to identify and target highly recurrent alterations in specific cancer contexts, such as EGFR mutations in non-small cell lung cancer (Lynch et al, N Engl J Med 350:2129-2139, 2004; Sharifnia et al., Proc Natl Acad Sci U S A 111:18661-18666, 2014), BCR-ABL translocations in chronic myeloid leukemia (Deininger, Pharmacol Rev 55:401-423. https://doi.org/10.1124/pr.55.3.4 , 2003; Druker et al, N Engl J Med 344. 1038-1042, 2001; Druker et al, N Engl J Med 344:1031-1037. https://doi.org/10.1056/NEJM200104053441401 , 2001), or HER2 amplifications in breast cancer (Slamon et al, N Engl J Med 344:783-792. https://doi.org/10.1056/NEJM200103153441101 , 2001; Solca et al, Beyond trastuzumab: second-generation targeted therapies for HER-2-positive breast cancer. In: Sibilia M, Zielinski CC, Bartsch R, Grunt TW (eds) Drugs for HER-2-positive breast cancer. Springer, Basel, pp 91-107, 2011). Despite these advances in our capacity to identify the genetic alterations that drive tumor initiation, survival, and proliferation, our ability to target these alterations to provide effective treatment options for patients in need, particularly those with rare or advanced cancers, remains limited (Gould et al, Nat Med 21:431-439. https://doi.org/10.1038/nm.3853 , 2015). Patient-derived models of cancer offer one potential mechanism to overcome this barrier between the bench and bedside. Through the development and testing of patient-derived models of cancer, functional genomics efforts can identify tumor-specific drug sensitivities and thereby provide a connection between tumor genetics and effective therapeutics for patients in need of treatment options.Recognizing that cancer is a multifaceted set of disease states, the development of personalized models of cancer that can be used to compare treatment options, identify tumor-specific vulnerabilities, and guide clinical decision-making has tremendous potential for improving patient outcomes. This chapter will describe a representative set of patient-derived models of cancer, reviewing each of their strengths and weaknesses and highlighting how selecting a model to suit a specific question or context is critical. Each model comes with a unique set of pros and cons, making them more or less appropriate for each specific research or clinical question. As each model can be leveraged to gain new insights into cancer biology, the key to their deployment is to identify the most appropriate model for a specific context, while carefully considering the strengths and limitations of the selected model. When used appropriately, patient-derived models may prove to be the missing link needed to bring the promise of personalized oncology to fruition in the clinic.

A Functional Precision Oncology Approach to Identify Treatment Strategies for Myxofibrosarcoma Patients.

In this era of precision medicine, numerous workflows for the targeting of high-recurrent mutations in common tumor types have been developed, leaving patients with rare diseases with few options. Here, we implement a functional precision oncology approach utilizing comprehensive genomic profiling in combination with high-throughput drug screening, to identify tumor-specific drug sensitivities for patients with rare tumor types such as myxofibrosarcoma. From a patient with a high-grade myxofibrosarcoma, who was enrolled in the Englander Institute for Precision Medicine (EIPM) program, we established patient-derived 3D sarco-spheres and xenograft models for functional testing. In the absence of a large cohort of clinically similar cases, high-throughput drug screening was performed on the patient-derived cells, and compared with two other myxofibrosarcoma lines and a benign fibroblast line to functionally identify tumor-specific drug sensitivities. The addition of functional drug sensitivity testing to complement genomic profiling identified multiple therapeutic options that were further validated in patient derived xenograft models. Genomic analyses detected the frequently known codeletion of the tumor suppressors CDKN2A/B together with the methylthioadenosine phosphorylase (MTAP) and a TP53 E286fs*50 mutation. High-throughput drug screening demonstrated tumor-specific sensitivity to compounds targeting the cell cycle. Based on genomic analysis and high-throughput drug screening, we show that targeting the cell cycle in these tumors is a powerful approach. IMPLICATIONS: This study demonstrates the potential of functional testing to aid clinical decision making for patients with rare or molecularly complex malignancies when combined with comprehensive genomic profiling.

Targeting Cullin-RING E3 Ubiquitin Ligase 4 by Small Molecule Modulators.

Cullin-RING E3 ubiquitin ligase 4 (CRL4) plays an essential role in cell cycle progression. Recent efforts using high throughput screening and follow up hit-to-lead studies have led to identification of small molecules 33-11 and KH-4-43 that inhibit E3 CRL4's core ligase complex and exhibit anticancer potential. This review provides: 1) an updated perspective of E3 CRL4, including structural organization, major substrate targets and role in cancer; 2) a discussion of the challenges and strategies for finding the CRL inhibitor; and 3) a summary of the properties of the identified CRL4 inhibitors as well as a perspective on their potential utility to probe CRL4 biology and act as therapeutic agents.

Inhibitors of cullin-RING E3 ubiquitin ligase 4 with antitumor potential.

Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4's core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4's substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds' cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4-expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.

Precision Targeting of pten-Null Triple-Negative Breast Tumors Guided by Electrophilic Metabolite Sensing.

Off-target effects continue to impede disease interventions, particularly when targeting a specific protein within a family of similar proteins, such as kinase isoforms that play tumor-subtype-specific roles in cancers. Exploiting the specific electrophilic-metabolite-sensing capability of Akt3, versus moderate or no sensing, respectively, by Akt2 and Akt1, we describe a first-in-class functionally Akt3-selective covalent inhibitor [MK-H(F)NE], wherein the electrophilic core is derived from the native reactive lipid metabolite HNE. Mechanistic profiling and pathway interrogations point to retention of the metabolite's structure-as opposed to implicit electrophilicity-as being essential for biasing isoform preference, which we found translates to tumor-subtype specificity against pten-null triple-negative breast cancers (TNBCs). MK-H(F)NE further enables novel downstream target identification specific to Akt3-function in disease. In TNBC xenografts, MK-H(F)NE fares better than reversible pan-Akt-inhibitors and does not show commonly observed side-effects associated with Akt1-inhibition. Inhibitors derived from native-metabolite sensing are thus an enabling plan-of-action for unmasking kinase-isoform-biased molecular targets and tumor-subtype-specific interventions.

Insulin-PI3K signalling: an evolutionarily insulated metabolic driver of cancer.

Cancer is driven by incremental changes that accumulate, eventually leading to oncogenic transformation. Although genetic alterations dominate the way cancer biologists think about oncogenesis, growing evidence suggests that systemic factors (for example, insulin, oestrogen and inflammatory cytokines) and their intracellular pathways activate oncogenic signals and contribute to targetable phenotypes. Systemic factors can have a critical role in both tumour initiation and therapeutic responses as increasingly targeted and personalized therapeutic regimens are used to treat patients with cancer. The endocrine system controls cell growth and metabolism by providing extracellular cues that integrate systemic nutrient status with cellular activities such as proliferation and survival via the production of metabolites and hormones such as insulin. When insulin binds to its receptor, it initiates a sequence of phosphorylation events that lead to activation of the catalytic activity of phosphoinositide 3-kinase (PI3K), a lipid kinase that coordinates the intake and utilization of glucose, and mTOR, a kinase downstream of PI3K that stimulates transcription and translation. When chronically activated, the PI3K pathway can drive malignant transformation. Here, we discuss the insulin-PI3K signalling cascade and emphasize its roles in normal cells (including coordinating cell metabolism and growth), highlighting the features of this network that make it ideal for co-option by cancer cells. Furthermore, we discuss how this signalling network can affect therapeutic responses and how novel metabolic-based strategies might enhance treatment efficacy for cancer.

Obesity-associated Adipose Stromal Cells Promote Breast Cancer Invasion Through Direct Cell Contact and ECM Remodeling.

Obesity increases the risk and worsens the prognosis for breast cancer due, in part, to altered adipose stromal cell (ASC) behavior. Whether ASCs from obese individuals increase migration of breast cancer cells relative to their lean counterparts, however, remains unclear. To test this connection, multicellular spheroids composed of MCF10A-derived tumor cell lines of varying malignant potential and lean or obese ASCs were embedded into collagen scaffolds mimicking the elastic moduli of interstitial breast adipose tissue. Confocal image analysis suggests that tumor cells alone migrate insignificantly under these conditions. However, direct cell-cell contact with either lean or obese ASCs enables them to migrate collectively, whereby obese ASCs activate tumor cell migration more effectively than their lean counterparts. Time-resolved optical coherence tomography (OCT) imaging suggests that obese ASCs facilitate tumor cell migration by mediating contraction of local collagen fibers. Matrix metalloproteinase (MMP)-dependent proteolytic activity significantly contributes to ASC-mediated tumor cell invasion and collagen deformation. However, ASC contractility is also important, as co-inhibition of both MMPs and contractility is necessary to completely abrogate ASC-mediated tumor cell migration. These findings imply that obesity-mediated changes of ASC phenotype may impact tumor cell migration and invasion with potential implications for breast cancer malignancy in obese patients.

PIP4Ks Suppress Insulin Signaling through a Catalytic-Independent Mechanism.

Insulin stimulates the conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), which mediates downstream cellular responses. PI(4,5)P2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks (PIP4K2A, PIP4K2B, and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P2 and a concomitant increase in insulin-stimulated production of PI(3,4,5)P3. The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P2 levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P2 levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLΦPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P2 synthesis and insulin-dependent conversion to PI(3,4,5)P3 and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling.

Rac-Mediated Macropinocytosis of Extracellular Protein Promotes Glucose Independence in Non-Small Cell Lung Cancer.

Cancer cells can adapt to nutrient poor conditions by rewiring their metabolism and using alternate fuel sources. Identifying these adaptive metabolic pathways may provide novel targets for cancer therapy. Here, we identify a subset of non-small cell lung cancer (NSCLC) cell lines that survive in the absence of glucose by internalizing and metabolizing extracellular protein via macropinocytosis. Macropinocytosis is increased in these glucose independent cells, and is regulated by phosphoinositide 3-kinase (PI3K) activation of Rac-Pak signaling. Furthermore, inhibition of Rac-dependent macropinocytosis blocks glucose-independent proliferation. We find that degradation of internalized protein produces amino acids, including alanine, which generates TCA cycle and glycolytic intermediates in the absence of glucose. In this process, the conversion of alanine to pyruvate by alanine transaminase 2 (ALT2) is critical for survival during glucose starvation. Collectively, Rac driven macropinocytosis of extracellular protein is an adaptive metabolic pathway used by a subset of lung cancers to survive states of glucose deprivation, and may serve as a potential drug target for cancer therapy.

Publisher Correction: Suppression of insulin feedback enhances the efficacy of PI3K inhibitors.

In this Letter, author Xing Du was incorrectly listed as Du Xing; this has been corrected online.

Suppression of insulin feedback enhances the efficacy of PI3K inhibitors.

Mutations in PIK3CA, which encodes the p110α subunit of the insulin-activated phosphatidylinositol-3 kinase (PI3K), and loss of function mutations in PTEN, which encodes a phosphatase that degrades the phosphoinositide lipids generated by PI3K, are among the most frequent events in human cancers1,2. However, pharmacological inhibition of PI3K has resulted in variable clinical responses, raising the possibility of an inherent mechanism of resistance to treatment. As p110α mediates virtually all cellular responses to insulin, targeted inhibition of this enzyme disrupts glucose metabolism in multiple tissues. For example, blocking insulin signalling promotes glycogen breakdown in the liver and prevents glucose uptake in the skeletal muscle and adipose tissue, resulting in transient hyperglycaemia within a few hours of PI3K inhibition. The effect is usually transient because compensatory insulin release from the pancreas (insulin feedback) restores normal glucose homeostasis3. However, the hyperglycaemia may be exacerbated or prolonged in patients with any degree of insulin resistance and, in these cases, necessitates discontinuation of therapy3-6. We hypothesized that insulin feedback induced by PI3K inhibitors may reactivate the PI3K-mTOR signalling axis in tumours, thereby compromising treatment effectiveness7,8. Here we show, in several model tumours in mice, that systemic glucose-insulin feedback caused by targeted inhibition of this pathway is sufficient to activate PI3K signalling, even in the presence of PI3K inhibitors. This insulin feedback can be prevented using dietary or pharmaceutical approaches, which greatly enhance the efficacy/toxicity ratios of PI3K inhibitors. These findings have direct clinical implications for the multiple p110α inhibitors that are in clinical trials and provide a way to increase treatment efficacy for patients with many types of tumour.

Cancer metabolism gets physical.

Patient-derived culture models enable assessment of drug sensitivity and can connect personalized genomics with therapeutic options. However, their clinical translation is constrained by limited fidelity. We outline how the physical microenvironment regulates cell metabolism and describe how engineered culture systems could enhance the predictive power for precision medicine.