bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022‒05‒15
24 papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. FASEB J. 2022 May;36 Suppl 1
      The conserved kinase mTOR (mechanistic target of rapamycin) regulates cell metabolism and promotes cell growth, proliferation, and survival in response to diverse environmental cues (e.g., nutrients; growth factors; hormones). mTOR forms the catalytic core of two multiprotein complexes, mTORC1 and mTORC2, which possess unique downstream targets and cellular functions. While mTORC1 and mTORC2 often respond to distinct upstream cues, they share a requirement for PI3K in their activation by growth factors. While many studies agree that amino acids activate mTORC1 but not mTORC2, several studies reported paradoxical activation of mTORC2 by amino acids. We noted that stimulating amino acid starved cells with a commercial mixture of amino acids increased mTORC2-dependent Akt S473 phosphorylation rapidly while re-feeding cells with complete DMEM containing amino acids failed to do so. Interestingly, we found the pH of the commercial amino acid mixture to be ~ pH 10. Upon controlling for pH, stimulating starved cells with amino acids at pH 10 but not 7.4 increased mTORC2 signaling. Moreover, DMEM at alkaline pH was sufficient to increase mTORC2 catalytic activity and signaling. Using a fluorescent pH-sensitive dye (cSNARF-1-AM) coupled to ratio-metric live cell imaging, we confirmed that alkaline extracellular pH (pHe) translated into a rapid increase in intracellular pH (pHi). Moreover, blunting this increase with a pharmacological inhibitor of an H+ transporter attenuated the increase in mTORC2 signaling by pHe. Alkaline pHi also activated AMPK, a canonical sensor of energetic stress that promotes mTORC2 signaling, as reported previously by us. Functionally, we found that alkaline pHi attenuated apoptosis caused by growth factor withdrawal through activation of AMPK-mTORC2 signaling. These results indicate that alkaline pHi augments mTORC2 signaling to promote cell survival, in part through AMPK. In the course of this work, we noted that pHi increased phosphorylation of several downstream targets of PI3K (e.g., Akt P-T308 and P-S473; S6K1 P-T389 and P-T229; PRAS40 P-T246; Tsc2 P-S939), suggesting that PI3K itself responds to changes in pHi. Indeed, alkaline pHi increased PI-3',4',5'-P3 levels in a manner sensitive to the PI3K inhibitor BYL-719. Thus, alkaline pHi elevates PI3K activity, which increases both mTORC1 and mTORC2 signaling. Mechanistically, we found that activation of PI3K by alkaline pHi induced dissociation of Tsc2 from lysosomal membranes, thereby relieving TSC-mediated suppression of Rheb, a mTORC1-activating GTPase. Functionally, we found that activation of PI3K by alkaline pHi increased mTORC1-mediated 4EBP1 phosphorylation, which initiates cap-dependent translation by eIF4E. Alkaline pHi also increased mTORC1-driven protein synthesis. Taken together, these findings reveal alkaline pHi as a previously unrecognized activator of PI3K-mTORC1/2 signaling that promotes protein synthesis and cell survival. As elevated pHi represents an under-appreciated hallmark of cancer cells, these findings suggest that by alkaline pHi sensing by the PI3K-mTOR axis and AMPK-mTORC2 axes may contribute to tumorigenesis.
  2. FASEB J. 2022 May;36 Suppl 1
      PIK3CA, the gene for the lipid kinase p110α is one of the most frequently mutated oncogenes across all types of cancer. p110α is an enzyme that catalyzes the formation of phosphatidylinositol 3, 4, 5 Triphosphate (PIP3 ). PIP3 recruits effector proteins which regulate growth, proliferation and motility. Due to this role as a master cell regulator, the activity of p110α is maintained in an inactive confirmation and is only activated downstream of Receptor Tyrosine Kinases (RTKs) and RAS family of GTPases. p110α is maintained in an inactive confirmation through interactions with its regulatory subunit as well as inhibitory contacts with the C-terminus. However, oncogenic mutations in p110α breaks these inhibitory interactions and drives hyperactivity without the need for activation signals leading to uncontrolled cell growth. We provide molecular insights into the regulation of oncogenic mutants of p110α using Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS). HDX-MS reveals the dynamic changes between the natural cytosolic state and fully-active membrane bound states of the enzyme. We find unique molecular mechanisms regulating how mutants at the c-terminus activate lipid kinase activity (H1047R, M1043L, G1049R and N1068KLKR). Using extensive biophysical tools, biochemical assays, and MD simulations, we have tested the oncogenic potential of these mutations. Our results elucidates a unifying theory towards the regulation of PI3Kα and how oncogenic mutations drive hyperactivity. This will aid in understanding the regulation of PI3Kα and in developing isoform specific inhibitors.
  3. FASEB J. 2022 May;36 Suppl 1
      The PI 3-kinase (PI3K) and AKT signaling pathway plays a critical role in regulating all aspects of normal cellular physiology, and is also frequently deregulated in human pathophysiologies, most evidently in cancer and diabetes. Growth factors and hormones stimulate PI3K leading to the biosynthesis of the lipid-derived second messenger PIP3. In turn, PIP3 elicits the membrane recruitment of the protein kinase AKT, originally discovered in 1987 by Staal and colleagues as v-Akt, a transforming oncogene. In the early 1990s, three independent groups cloned and described the cellular homolog c-AKT, a serine/threonine protein kinase with a high degree of homology to other AGC family protein kinases. In the ensuing three decades, the mechanisms by which AKT transduces signals to cell growth, proliferation, motility and metabolism were uncovered. Three AKT isoforms exist in humans encoded by distinct genes (AKT1, AKT2, AKT3), and although originally thought to function redundantly, many studies have shown that AKT isoforms have non-overlapping and unique roles in both normal physiology and disease. Similarly, genetic lesions in the PI3K and AKT oncogenes have been described, and many of the genes that contribute to PI3K/AKT pathway activation and also signal termination have been found to be altered in human cancers. Numerous drugs that inhibit PI3K as well AKT have been developed for therapeutic use in patients, and many of these are being evaluated in late-stage clinical trials. During the lecture, I will highlight the major advances in PI3K and AKT field over the past 30 years, with a focus on mechanistic insight into this ubiquitous lipid signaling pathway. Genetic lesions in the PI3K/AKT pathway in human cancers will also be discussed, as well as efforts to target this pathway therapeutically. The second part of the lecture will focus on recent efforts in our laboratory to uncover novel mechanisms of AKT signaling and biology, with an emphasis on breast cancer and with a focus on metabolic reprogramming mediated by AKT. I will also present recent efforts aimed at targeting AKT with novel therapies, including degrader technologies and how these have illuminated novel aspects of AKT biology. I will conclude with some personal thoughts and future perspectives as to where the field is going, gaps in knowledge and what studying AKT for 30 years has taught me.
  4. Curr Protoc. 2022 May;2(5): e416
      The serine/threonine protein kinase Akt integrates diverse upstream inputs to regulate cell survival, growth, metabolism, migration, and differentiation. Mounting evidence suggests that Akt activity is differentially regulated depending on its subcellular location, which can include the plasma membrane, endomembrane, and nuclear compartment. This spatial control of Akt activity is critical for achieving signaling specificity and proper physiological functions, and deregulation of compartment-specific Akt signaling is implicated in various diseases, including cancer and diabetes. Understanding the spatial coordination of the signaling network centered around this key kinase and the underlying regulatory mechanisms requires precise tracking of Akt activity at distinct subcellular compartments within its native biological contexts. To address this challenge, new molecular tools are being developed, enabling us to directly interrogate the spatiotemporal regulation of Akt in living cells. These include, for instance, the newly developed genetically encodable fluorescent-protein-based Akt kinase activity reporter (AktAR2), which serves as a substrate surrogate of Akt kinase and translates Akt-specific phosphorylation into a quantifiable change in Förster resonance energy transfer (FRET). In addition, we developed the Akt substrate tandem occupancy peptide sponge (Akt-STOPS), which allows biochemical perturbation of subcellular Akt activity. Both molecular tools can be readily targeted to distinct subcellular localizations. Here, we describe a workflow to study Akt kinase activity at different subcellular locations in living cells. We provide a protocol for using genetically targeted AktAR2 and Akt-STOPS, along with fluorescence imaging in living NIH3T3 cells, to visualize and perturb, respectively, the activity of endogenous Akt kinase at different subcellular compartments. We further describe a protocol for using chemically inducible dimerization (CID) to control the plasma membrane-specific inhibition of Akt activity in real time. Lastly, we describe a protocol for maintaining NIH3T3 cells in culture, a cell line known to exhibit robust Akt activity. In all, this approach enables interrogation of spatiotemporal regulation and functions of Akt, as well as the intricate signaling networks in which it is embedded, at specific subcellular locations. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualizing and perturbing subcellular Akt kinase activity using AktAR and Akt-STOPS Basic Protocol 2: Using chemically inducible dimerization (CID) to control inhibition of Akt at the plasma membrane Support Protocol: Maintaining NIH3T3 cells in culture.
    Keywords:  compartmentalized signaling; fluorescence; live-cell imaging; location-specific; protein kinase B
  5. Orphanet J Rare Dis. 2022 May 07. 17(1): 189
      BACKGROUND: PROS disorders are driven by somatic, gain-of-function mutations in PIK3CA that result in hyperactivation of the phosphatidylinositol-3-kinase (PI3K) signaling pathway. PROS encompasses a broad spectrum of overlapping phenotypes (including overgrowth and vascular malformations) that vary significantly in their severity; every case is unique, leading to different, complex experiences. Here, we aim to describe the PROS experience from the patients' and caregivers' points of view, from onset to diagnosis to treatment and support.RESULTS: The PROS patient journey was developed using a literature review, an ethnography study, health care professional (HCP) research, and social listening. It was then validated with patients, caregivers, and patient advocates. Physician research included 94 PROS centers and other vascular anomaly centers throughout the United States and Europe. Ethnographic research included 24 patients, caregivers, and/or advocates; selected data from 223 patients were reviewed. Key priority areas of need were identified, along with barriers to and potential enablers of quality care. Visual mapping of the PROS patient and family journey was developed to identify key personal health and system issues, and opportunities for improvements throughout patients' lifespans. Maps were also developed for 3 specific conditions: Klippel-Trénaunay syndrome (K-T); congenital lipomatous overgrowth, vascular malformations, epidermal nevi, scoliosis/skeletal and spinal anomalies (CLOVES) syndrome; and megalencephaly-capillary malformation syndrome (M-CM). Overall, most patients with PROS conditions and their families struggle with a long path to diagnosis, access to genetic testing, and finding qualified specialists. Following diagnosis, patients and families are frequently challenged with major medical events, comorbidities, unpredictability, frequent hospitalization, impact on school and work, the need for multidisciplinary care, unwanted attention, adverse impact on mental and emotional health, and financial pressures. Lack of effective pain management emerged as a substantial issue. Challenges and barriers to quality care shift throughout patients' lifespans; transition from pediatric to adult care can be especially difficult.
    CONCLUSIONS: This patient journey in PROS was created in collaboration with patients, caregivers, and advocates as key partners. This novel methodology, which could be applied elsewhere, can more accurately identify areas of unmet need, barriers to care, education topics, and assist HCPs to understand the patient and family perspective.
    Keywords:  PI3K; PIK3CA; PROS; Vascular malformation
  6. Elife. 2022 May 12. pii: e76183. [Epub ahead of print]11
      Tyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time- and space-dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer, knocking down which attenuates metastatic phenotypes. However, due to the limited number of known substrates in the ovarian cancer context, the molecular basis for its pro-proliferation activity remains enigmatic. Here, we employed mass spectrometry and biochemical approaches to identify insulin receptor substrate 4 (IRS4) as a novel substrate of FER. FER engaged its kinase domain to associate with the PH and PTB domains of IRS4. Using a proximity-based tagging system in ovarian carcinoma-derived OVCAR-5 cells, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85β, the regulatory subunit of PI3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4 delayed ovarian tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.
    Keywords:  E. coli; FER; IRS4; PIK3R2; biochemistry; cancer biology; chemical biology; human; mouse; ovarian cancer; tyrosine phosphorylation
  7. Int J Mol Sci. 2022 Apr 27. pii: 4840. [Epub ahead of print]23(9):
      As integral parts of pathological arterial thrombi, platelets are the targets of pharmacological regimens designed to treat and prevent thrombosis. A detailed understanding of platelet biology and function is thus key to design treatments that prevent thrombotic cardiovascular disease without significant disruption of the haemostatic balance. Phosphoinositide 3-kinases (PI3Ks) are a group of lipid kinases critical to various aspects of platelet biology. There are eight PI3K isoforms, grouped into three classes. Our understanding of PI3K biology has recently progressed with the targeting of specific isoforms emerging as an attractive therapeutic strategy in various human diseases, including for thrombosis. This review will focus on the role of PI3K subtypes in platelet function and subsequent thrombus formation. Understanding the mechanisms by which platelet function is regulated by the various PI3Ks edges us closer toward targeting specific PI3K isoforms for anti-thrombotic therapy.
    Keywords:  PI3K; antiplatelet therapy; phosphoinositide 3-kinase; platelets; thrombosis
  8. Oncogene. 2022 May 10.
      p110α is a catalytic subunit of phosphoinositide 3-kinase (PI3K), a major downstream effector of receptor tyrosine kinase ErbB2, that is amplified and overexpressed in 20-30% of breast cancers, 40% of which have an activating mutation in p110α. Despite the high frequency of PIK3CA gain-of-function mutations, their prognostic value is controversial. Here, we employ a knock-in transgenic strategy to restrict the expression of an activated form of ErbB2 and p110α kinase domain mutation (p110αHR) in the mammary epithelium. Physiological levels of transgene expression under the control of their endogenous promoters did not result in a major synergistic effect. However, tumors arising in ErbB2/p110αHR bi-genic strain metastasized to the lung with significantly reduced capacity compared to tumors expressing ErbB2 alone. The reduced metastasis was further associated with retention of the myoepithelial layer reminiscent of ductal carcinoma in situ (DCIS), a non-invasive stage of human breast cancer. Molecular and biochemical analyses revealed that these poorly metastatic tumors exhibited a significant decrease in phospho-myosin light chain 2 (MLC2) associated with cellular contractility and migration. Examination of human samples for MLC2 activity revealed a progressive increase in cellular contractility between non-invasive DCIS and invasive ductal carcinoma. Collectively, these data argue that p110αHR mutation attenuates metastatic behavior in the context of ErbB2-driven breast cancer.
  9. Science. 2022 May 13. 376(6594): 695-696
      Single-cell analyses reveal tissue-agnostic features and tissue-specific cell states.
  10. FASEB J. 2022 May;36 Suppl 1
      The blood-brain barrier (BBB) is a specialized microvasculature integral for brain tissue-fluid homeostasis that is comprised of brain endothelial cells (BECs) and pericytes. Disruption of the BBB and subsequent vascular leakage of protein-rich fluids is toxic to surrounding neurons and is an underlying risk factor in neurodegenerative disorders. BECs and pericytes form adhesions through the transmembrane protein Neural (N)-cadherin. Although BEC-pericyte interactions are critical for maintaining the BBB, the role of N-cadherin adhesions in regulating the BBB remain unclear. Our previous work demonstrated that mutant mice lacking Cdh2 (N-cadherin) in ECs or pericytes exhibited a size-dependent increase in BBB permeability without affecting vessel or pericyte coverage. These findings raise the possibility that N-cadherin junctions activate outside-in signaling to strengthen the BBB. Analysis of tight junction (TJ) proteins occludin and claudins 1 and 5 in microvascular BECs of the cortex demonstrated a significant reduction in the accumulation of occludin, but not claudins, at TJs of KO mice. To delineate the signaling mechanism by which N-cadherin adhesion-mediated signaling regulates occludin TJs, we utilize biomimetic surfaces (Ncdh-BioS) bearing covalently linked N-cadherin extracellular domain to induce N-cadherin adhesion in BEC monolayers in vitro. Consistent with our observations in mice, assembly of N-cadherin junctions induced the accumulation of occludin and ZO1 at TJs in BEC monolayers. Depletion of N-cadherin reversed these events, suggesting that N-cadherin adhesion-induced signaling assembles or stabilizes occludin TJs. Analysis of occludin-Dendra 2 kinetics revealed decreased internalization rates of occludin from TJs in BECs grown on Ncdh-BioS as compared to collagen, suggesting that N-cadherin signaling stabilized occludin TJs. Furthermore, formation of N-cadherin adhesion complexes led to activation of phosphoinositide 3-kinase (PI3K) signaling as evidenced by the spatial redistribution of Akt to the plasma membrane as well as Akt phosphorylation. Pharmacological inhibition of class I PI3K with Copanlisib or depletion of PI3K p110β, but not of other PI3K catalytic isoforms, abolished Akt activation and increased the rate of occludin internalization from TJs in BECs grown on Ncdh-BioS. Cumulatively, these data demonstrates that N-cadherin outside-in signaling strengthens the BBB by stabilizing occludin TJs through PI3K p110β signaling.
  11. FASEB J. 2022 May;36 Suppl 1
      mTOR, which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA binding protein, AUF1 (ARE/poly(U)-binding/degradation factor 1), modulates mTORC2/Akt signaling. AUF1 is required for Akt phosphorylation. It also mediates phosphorylation of the mTORC2-modulated metabolic enzyme GFAT1 at Ser243. Reciprocally, mTORC2 could also modulate AUF1. Conditions that enhance mTORC2 signaling, such as serum restimulation or acute glutamine withdrawal augments AUF1 phosphorylation while mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in mTORC2/Akt signaling. Targeting AUF1 could serve as a novel treatment strategy for cancers with upregulated mTORC2/Akt signaling.
  12. Proc Natl Acad Sci U S A. 2022 May 17. 119(20): e2123261119
      SignificanceThe mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is frequently elevated in human disease, including cancer, type 2 diabetes, metabolic disorders, and neurodegeneration. We identify SNAT7 as an important regulator of mTORC1. We believe this research will provide valuable insight about mTORC1 biology and may uncover novel therapeutic targets for patients.
    Keywords:  SNAT7; mTOR; macropinocytosis
  13. iScience. 2022 Mar 18. 25(3): 103944
      Patient heterogeneity precludes cancer treatment and drug development; hence, development of methods for finding prognostic markers for individual treatment is urgently required. Here, we present Pasmopy (Patient-Specific Modeling in Python), a computational framework for stratification of patients using in silico signaling dynamics. Pasmopy converts texts and sentences on biochemical systems into an executable mathematical model. Using this framework, we built a model of the ErbB receptor signaling network, trained in cultured cell lines, and performed in silico simulation of 377 patients with breast cancer using The Cancer Genome Atlas (TCGA) transcriptome datasets. The temporal dynamics of Akt, extracellular signal-regulated kinase (ERK), and c-Myc in each patient were able to accurately predict the difference in prognosis and sensitivity to kinase inhibitors in triple-negative breast cancer (TNBC). Our model applies to any type of signaling network and facilitates the network-based use of prognostic markers and prediction of drug response.
    Keywords:  Cancer; Drugs; Molecular network; Systems biology
  14. Nature. 2022 May 12.
      DddA-derived cytosine base editors (DdCBEs), which are fusions of the split-DddA halves and transcription activator-like effector (TALE) array proteins, enable targeted C·G-to- T·A conversions in mitochondrial DNA1. However, its genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or -independent. TAS-dependent off-target sites in the nuclear DNA (nDNA) are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by a paired TALE proteins positioned in close proximity. TAS-independent nDNA off-target sites are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with CTCF-binding sites and are enriched in TAD boundaries. We also engineered DdCBE to alleviate such off-target effect. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base editing tools.
  15. FASEB J. 2022 May;36 Suppl 1
      Akt1 and Akt2 are the main protein kinase B (Akt) isoforms expressed in the mammalian heart. Tamoxifen(OHTx) -inducible, cardiomyocyte-specific Akt1/ Akt2 double knockout mice (iCM-Akt12) show progressive cardiac atrophy and loss of contractile function leading to terminal heart failure 23.9 ± 2 days after first OHTx injection. TUNEL staining of iCM-Akt12 hearts revealed that cardiomyocyte apoptosis did not contribute substantially to cardiac atrophy. Rather, cellular atrophy caused the loss of cardiac mass. The cellular area (a), width (w), and length (l) declined between day 9 and day 14 [-18 (a)/-18 (w)/-9% (l)] as compared to WT controls. This size reduction was retarded between d14 and d21 (-20 (a)/-24 (w)/-8% (l). In vivo 31 P-imaging identified a progressive energetic deficiency of iCM-Akt12 hearts on d15 and d20 after OHTx injection. Further analyses showed that in the early phase up to day 14 increased autophagic activity and reduced de novo protein synthesis seemed to cause cellular atrophy. As expected, deletion of Akt led to impaired mTORC1 signaling indicated by reduced phosphorylation of S6K, RPS6, 4E-BP1, all involved in translation. Concomitantly, we measured reduced protein synthesis. Autophagy was increased in iCM-Akt12 hearts, as shown by increased LC3-II/I ratio and higher expression of autophagic genes. From day 14 after KO induction (late phase), protein synthesis appeared to increase and mTORC1 substrates involved in translation (S6K, RPS6 and 4E-BP1) were higher phosphorylated. Of note, also a higher phosphorylation of the inhibitory mTORC1 target site in the central autophagy kinase Ulk1 (S757) was found. Moreover, autophagy appeared to be disturbed, as p62 accumulated. Despite increasing energetic depletion, the cellular energy sensor AMPK was not activated in iCM-Akt12 hearts. Rather, AMPK was inhibited by phosphorylation of the α-subunit on serine 485/491. The S485/491 phosphorylation has been attributed to several kinases (Akt, PKD1, S6K, PKC, PKA). Akt deletion led to a pronounced hyperactivation of Pdk1 signaling upstream of Akt, causing an activation of protein kinases regulated by Pdk1 (S6K, PKA, PKC, PKD). This was demonstrated by increased phosphorylation of specific kinase substrates and consensus motifs in late iCM-Akt12 hearts and might promote the AMPK inactivation. To test to what extent a non-inhibitable AMPK might attenuate the fatal phenotype of cardiac Akt loss, AAV-mediated infection of iCM-Akt12 mice with a phospho-defective AMPKα2 S491A mutant prior to KO induction was performed. This led to an increased survival and improved heart function, demonstrating that the AMPK inhibition contributes to the lethal phenotype. In conclusion, loss of Akt signaling leads to a widespread dysregulation of intracellular signal transduction. Whereas enhanced autophagy and inhibition of mTORC1 are direct consequences of Akt deletion, the later reactivation of mTORC1 and inhibition of AMPK causes attenuation of autophagy, increased translation, deceleration of cellular atrophy, worsenes energetic shortage, and aggravates cardiac dysfunction.
  16. FASEB J. 2022 May;36 Suppl 1
      The Extracellular signal-regulated kinase (ERK) signaling axis is activated by a host of divergent signaling molecules which each induce their own specific responses. These cellular responses include fundamental processes such as proliferation, differentiation, migration, and, under certain conditions, even apoptosis. Thus, the ubiquitous utilization of ERK signaling in variable and contrasting cellular processes makes it difficult to inhibit the pathway without causing adverse side effects. Furthermore, dysregulation of the Ras/Raf/MEK/ERK signaling pathway is directly implicated in a variety of disorders ranging from cancer to diabetes. Here, we report new tools to elucidate the nuanced mechanisms of differential regulation of the ERK pathway. First, we report a new genetically-encodable tool to inhibit ERK at specific subcellular locations, allowing us to explore the specific function and mechanisms of regulation of ERK with extremely precise subcellular resolution. Additionally, we report a new genetically-encodable fluorescent biosensor based on previous generations of the ERK-kinase activity reporter (EKAR). Our new biosensor, which we have termed reDox-EKAR1, allows us to probe into the spatiotemporal activity of ERK towards a different class of substrates than substrates probed by previous generations of EKAR. Utilizing these new tools, we report how specific pools of ERK regulate particular cellular functions previously underappreciated in the field, and we present our preliminary results detailing how these specific subcellular pools of ERK are differentially regulated.
  17. Cancer Cell. 2022 May 06. pii: S1535-6108(22)00176-3. [Epub ahead of print]
      Animal models have evolved to be a key component of translational research of cancer, and they are now extensively used to test drug candidates, predict drug responses, and essentially drive discovery of cancer biology. However, the model-centric approach has not yielded the expected abundance of treatment advances. We propose that the focus of translational research needs to shift from animal models to human patients, and the goal should be to understand why tumor responses and outcomes are so variable between patients and how it can be predicted at the individual level and thus to generate hypotheses that are more relevant to people than to atypically sensitive animal models.
  18. PLoS Comput Biol. 2022 May 13. 18(5): e1010110
      Phosphoproteomic experiments routinely observe thousands of phosphorylation sites. To understand the intracellular signaling processes that generated this data, one or more causal protein kinases must be assigned to each phosphosite. However, limited knowledge of kinase specificity typically restricts assignments to a small subset of a kinome. Starting from a statistical model of a high-throughput, in vitro kinase-substrate assay, I have developed an approach to high-coverage, multi-label kinase-substrate assignment called IV-KAPhE ("In vivo-Kinase Assignment for Phosphorylation Evidence"). Tested on human data, IV-KAPhE outperforms other methods of similar scope. Such computational methods generally predict a densely connected kinase-substrate network, with most sites targeted by multiple kinases, pointing either to unaccounted-for biochemical constraints or significant cross-talk and signaling redundancy. I show that such predictions can potentially identify biased kinase-site misannotations within families of closely related kinase isozymes and they provide a robust basis for kinase activity analysis.
  19. Nat Metab. 2022 May 09.
      Tumorigenesis is associated with elevated glucose and glutamine consumption, but how cancer cells can sense their levels to activate lipid synthesis is unknown. Here, we reveal that ammonia, released from glutamine, promotes lipogenesis via activation of sterol regulatory element-binding proteins (SREBPs), endoplasmic reticulum-bound transcription factors that play a central role in lipid metabolism. Ammonia activates the dissociation of glucose-regulated, N-glycosylated SREBP-cleavage-activating protein (SCAP) from insulin-inducible gene protein (Insig), an endoplasmic reticulum-retention protein, leading to SREBP translocation and lipogenic gene expression. Notably, 25-hydroxycholesterol blocks ammonia to access its binding site on SCAP. Mutating aspartate D428 to alanine prevents ammonia binding to SCAP, abolishes SREBP-1 activation and suppresses tumour growth. Our study characterizes the unknown role, opposite to sterols, of ammonia as a key activator that stimulates SCAP-Insig dissociation and SREBP-1 activation to promote tumour growth and demonstrates that SCAP is a critical sensor of glutamine, glucose and sterol levels to precisely control lipid synthesis.
  20. Nature. 2022 May 11.
      Missense driver mutations in cancer are concentrated in a few hotspots1. Various mechanisms have been proposed to explain this skew, including biased mutational processes2, phenotypic differences3-6 and immunoediting of neoantigens7,8; however, to our knowledge, no existing model weighs the relative contribution of these features to tumour evolution. We propose a unified theoretical 'free fitness' framework that parsimoniously integrates multimodal genomic, epigenetic, transcriptomic and proteomic data into a biophysical model of the rate-limiting processes underlying the fitness advantage conferred on cancer cells by driver gene mutations. Focusing on TP53, the most mutated gene in cancer1, we present an inference of mutant p53 concentration and demonstrate that TP53 hotspot mutations optimally solve an evolutionary trade-off between oncogenic potential and neoantigen immunogenicity. Our model anticipates patient survival in The Cancer Genome Atlas and patients with lung cancer treated with immunotherapy as well as the age of tumour onset in germline carriers of TP53 variants. The predicted differential immunogenicity between hotspot mutations was validated experimentally in patients with cancer and in a unique large dataset of healthy individuals. Our data indicate that immune selective pressure on TP53 mutations has a smaller role in non-cancerous lesions than in tumours, suggesting that targeted immunotherapy may offer an early prophylactic opportunity for the former. Determining the relative contribution of immunogenicity and oncogenic function to the selective advantage of hotspot mutations thus has important implications for both precision immunotherapies and our understanding of tumour evolution.
  21. FASEB J. 2022 May;36 Suppl 1
      The function of MAPK phosphatase-2 (MKP-2), a type 1 dual-specific phosphatase (DUSP) in metabolic regulation is largely unknown. Here we demonstrate that MKP-2 expression was upregulated in liver tissue in humans with obesity and fatty liver disease, and in insulin-responsive tissues in mice with obesity. MKP-2 deficient mice have enhanced p38 MAPK, JNK and ERK activities in insulin-responsive tissues compared with wild type mice. MKP-2 deficiency in mouse protects against diet-induced obesity and hepatic steatosis and was accompanied with improved glucose homeostasis and insulin sensitivity. This was associated with enhanced circulating insulin-like growth factor-1 (IGF-1) and stromal cell-derived factor 1 (SDF-1) levels in Mkp-2-/- mice. PTEN, a negative regulator of Akt, was upregulated in livers of Mkp-2-/- mice, resulting in enhanced Akt activity consistent with increased insulin sensitivity. Pancreatic islet analysis demonstrated that MKP-2 deficiency altered islet composition and this has the potential to regulate in b-cell physiology. These studies demonstrate for the first time that MKP-2 is essential in the regulation of metabolic homeostasis and pathophysiology of obesity-induced insulin resistance and fatty liver disease.
  22. Cell. 2022 May 09. pii: S0092-8674(22)00472-X. [Epub ahead of print]
      Giant congenital melanocytic nevi are NRAS-driven proliferations that may cover up to 80% of the body surface. Their most dangerous consequence is progression to melanoma. This risk often triggers preemptive extensive surgical excisions in childhood, producing severe lifelong challenges. We have presented preclinical models, including multiple genetically engineered mice and xenografted human lesions, which enabled testing locally applied pharmacologic agents to avoid surgery. The murine models permitted the identification of proliferative versus senescent nevus phases and treatments targeting both. These nevi recapitulated the histologic and molecular features of human giant congenital nevi, including the risk of melanoma transformation. Cutaneously delivered MEK, PI3K, and c-KIT inhibitors or proinflammatory squaric acid dibutylester (SADBE) achieved major regressions. SADBE triggered innate immunity that ablated detectable nevocytes, fully prevented melanoma, and regressed human giant nevus xenografts. These findings reveal nevus mechanistic vulnerabilities and suggest opportunities for topical interventions that may alter the therapeutic options for children with congenital giant nevi.
    Keywords:  Nras; congenital melanocytic nevus; hapten; melanoma; mole; prevention; topical
  23. Commun Biol. 2022 May 11. 5(1): 438
      Multiplex imaging technologies are increasingly used for single-cell phenotyping and spatial characterization of tissues; however, transparent methods are needed for comparing the performance of platforms, protocols and analytical pipelines. We developed a python software, mplexable, for reproducible image processing and utilize Jupyter notebooks to share our optimization of signal removal, antibody specificity, background correction and batch normalization of the multiplex imaging with a focus on cyclic immunofluorescence (CyCIF). Our work both improves the CyCIF methodology and provides a framework for multiplexed image analytics that can be easily shared and reproduced.
  24. Blood. 2022 May 11. pii: blood.2021015106. [Epub ahead of print]
      Relapse and refractory T cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 inhibitors and the general tyrosine kinase-inhibitor, dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.