bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022‒02‒27
28 papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. Cancer Med. 2022 Feb 25.
      PURPOSE: The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway controls insulin sensitivity and glucose metabolism. Hyperglycemia is one of the most common on-target adverse effects (AEs) of PI3K/AKT inhibitors. As several PI3K and AKT inhibitors are approved by the United States Food and Drug Administration or are being studied in clinical trials, characterizing this AE and developing a management strategy is essential.METHODS: Patients with hematologic or solid malignancies treated at Memorial Sloan Kettering Cancer Center with a PI3K or AKT inhibitor were included in this retrospective analysis. A search for patients experiencing hyperglycemia was performed. The frequency, management interventions and outcomes were characterized.
    RESULTS: Four hundred and ninety-one patients with 10 unique cancer types who received a PI3K or AKT inhibitor were included. Twelve percent of patients required a dose interruption, 6% of patients required a dose reduction and 2% of patients were hospitalized to manage hyperglycemia. No events occurred among patients receiving β-, γ-, or δ- specific PI3K inhibitor. There was one case where the PI3K or AKT inhibitor was permanently discontinued due to hyperglycemia. Metformin was the most commonly used antidiabetic medication, followed by insulin, sodium-glucose transport protein 2 (SGLT2) inhibitors, and sulfonylurea. SGLT2 inhibitors were associated with the greatest reductions in blood sugar, followed by metformin. At least one case of euglycemic diabetic ketoacidosis (DKA) occurred in a patient on PI3K inhibitor and SGLT2 inhibitor. Body mass index ≥ 25 and HbA1c  ≥ 5.7 are were independently significant predictors of developing hyperglycemia.
    CONCLUSION: Hyperglycemia is one of the major on-target side effects of PI3K and AKT inhibitors. It is manageable with antidiabetic medications, treatment interruption and/or dose modification. We summarize pharmacological interventions that may be considered for PI3K/AKT inhibitor induced hyperglycemia. SGLT2-inhibitor may be a particularly effective second-line option after metformin but there is a low risk of euglycemic DKA, which can be deadly. To our knowledge, our report is the largest study of hyperglycemia in patients receiving PI3K/AKT inhibitors.
    Keywords:  PI3K/AKT inhibitors; SGLT2 inhibitors; hyperglycemia; risk factors; toxicity management
  2. Dev Cell. 2022 Feb 15. pii: S1534-5807(22)00070-3. [Epub ahead of print]
      The coordinated regulation of growth control and metabolic pathways is required to meet the energetic and biosynthetic demands associated with proliferation. Emerging evidence suggests that the Hippo pathway effector Yes-associated protein 1 (YAP) reprograms cellular metabolism to meet the anabolic demands of growth, although the mechanisms involved are poorly understood. Here, we demonstrate that YAP co-opts the sterol regulatory element-binding protein (SREBP)-dependent lipogenic program to facilitate proliferation and tissue growth. Mechanistically, YAP stimulates de novo lipogenesis via mechanistic target of rapamcyin (mTOR) complex 1 (mTORC1) signaling and subsequent activation of SREBP. Importantly, YAP-dependent regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1) is required to activate mTORC1/SREBP and stimulate de novo lipogenesis. We also find that the SREBP target genes fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) are conditionally required to support YAP-dependent proliferation and tissue growth. These studies reveal that de novo lipogenesis is a metabolic vulnerability that can be targeted to disrupt YAP-dependent proliferation and tissue growth.
    Keywords:  SGK1; SREBP; YAP; cell metabolism; growth; lipogenesis; mTORC1; proliferation
  3. Nature. 2022 Feb 23.
      Combinations of anti-cancer drugs can overcome resistance and provide new treatments1,2. The number of possible drug combinations vastly exceeds what could be tested clinically. Efforts to systematically identify active combinations and the tissues and molecular contexts in which they are most effective could accelerate the development of combination treatments. Here we evaluate the potency and efficacy of 2,025 clinically relevant two-drug combinations, generating a dataset encompassing 125 molecularly characterized breast, colorectal and pancreatic cancer cell lines. We show that synergy between drugs is rare and highly context-dependent, and that combinations of targeted agents are most likely to be synergistic. We incorporate multi-omic molecular features to identify combination biomarkers and specify synergistic drug combinations and their active contexts, including in basal-like breast cancer, and microsatellite-stable or KRAS-mutant colon cancer. Our results show that irinotecan and CHEK1 inhibition have synergistic effects in microsatellite-stable or KRAS-TP53 double-mutant colon cancer cells, leading to apoptosis and suppression of tumour xenograft growth. This study identifies clinically relevant effective drug combinations in distinct molecular subpopulations and is a resource to guide rational efforts to develop combinatorial drug treatments.
  4. Nat Rev Genet. 2022 Feb 25.
      The development of single-cell and spatial transcriptomics methods was instrumental in the conception of the Human Cell Atlas initiative, which aims to generate an integrated map of all cells across the human body. These technology advances are bringing increasing depth and resolution to maps of human organs and tissues, as well as our understanding of individual human cell types. Commonalities as well as tissue-specific features of primary and supportive cell types across human organs are beginning to emerge from these human tissue maps. In this Review, we highlight key biological insights obtained from cross-tissue studies into epithelial, fibroblast, vascular and immune cells based on single-cell gene expression data in humans and contrast it with mechanisms reported in mice.
  5. Elife. 2022 Feb 25. pii: e71994. [Epub ahead of print]11
      Quantifying the activity of gene expression signatures is common in analyses of single-cell RNA sequencing data. Methods originally developed for bulk samples are often used for this purpose without accounting for contextual differences between bulk and single-cell data. More broadly, these methods have not been benchmarked. Here we benchmark five such methods, including single sample gene set enrichment analysis (ssGSEA), Gene Set Variation Analysis (GSVA), AUCell, Single Cell Signature Explorer (SCSE), and a new method we developed, Jointly Assessing Signature Mean and Inferring Enrichment (JASMINE). Using cancer as an example, we show cancer cells consistently express more genes than normal cells. This imbalance leads to bias in performance by bulk-sample-based ssGSEA in gold standard tests and down sampling experiments. In contrast, single-cell-based methods are less susceptible. Our results suggest caution should be exercised when using bulk-sample-based methods in single-cell data analyses, and cellular contexts should be taken into consideration when designing benchmarking strategies.
    Keywords:  cancer biology; computational biology; human; systems biology
  6. Mol Cell. 2022 Feb 16. pii: S1097-2765(22)00105-8. [Epub ahead of print]
      Fumarate is an oncometabolite. However, the mechanism underlying fumarate-exerted tumorigenesis remains unclear. Here, utilizing human type2 papillary renal cell carcinoma (PRCC2) as a model, we show that fumarate accumulates in cells deficient in fumarate hydratase (FH) and inhibits PTEN to activate PI3K/AKT signaling. Mechanistically, fumarate directly reacts with PTEN at cysteine 211 (C211) to form S-(2-succino)-cysteine. Succinated C211 occludes tethering of PTEN with the cellular membrane, thereby diminishing its inhibitory effect on the PI3K/AKT pathway. Functionally, re-expressing wild-type FH or PTEN C211S phenocopies an AKT inhibitor in suppressing tumor growth and sensitizing PRCC2 to sunitinib. Analysis of clinical specimens indicates that PTEN C211 succination levels are positively correlated with AKT activation in PRCC2. Collectively, these findings elucidate a non-metabolic, oncogenic role of fumarate in PRCC2 via direct post-translational modification of PTEN and further reveal potential stratification strategies for patients with FH loss by combinatorial AKTi and sunitinib therapy.
    Keywords:  FH deficiency; PI3K/AKT; PRCC2; PTEN; TKI resistance; fumarate; succination; therapeutic resistance; tumorigenesis; type-2 papillary renal cell carcinoma
  7. Front Physiol. 2021 ;12 779121
      Insulin and IGF-1, acting through the insulin receptor (IR) and IGF-1 receptor (IGF1R), maintain muscle mass and mitochondrial function, at least part of which occurs via their action to regulate gene expression. Here, we show that while muscle-specific deletion of IR or IGF1R individually results in only modest changes in the muscle transcriptome, combined deletion of IR/IGF1R (MIGIRKO) altered > 3000 genes, including genes involved in mitochondrial dysfunction, fibrosis, cardiac hypertrophy, and pathways related to estrogen receptor, protein kinase A (PKA), and calcium signaling. Functionally, this was associated with decreased mitochondrial respiration and increased ROS production in MIGIRKO muscle. To determine the role of FoxOs in these changes, we performed RNA-Seq on mice with muscle-specific deletion of FoxO1/3/4 (M-FoxO TKO) or combined deletion of IR, IGF1R, and FoxO1/3/4 in a muscle quintuple knockout (M-QKO). This revealed that among IR/IGF1R regulated genes, >97% were FoxO-dependent, and their expression was normalized in M-FoxO TKO and M-QKO muscle. FoxO-dependent genes were related to oxidative phosphorylation, inflammatory signaling, and TCA cycle. Metabolomic analysis showed accumulation of TCA cycle metabolites in MIGIRKO, which was reversed in M-QKO muscle. Likewise, calcium signaling genes involved in PKA signaling and sarcoplasmic reticulum calcium homeostasis were markedly altered in MIGIRKO muscle but normalized in M-QKO. Thus, combined loss of insulin and IGF-1 action in muscle transcriptionally alters mitochondrial function and multiple regulatory and signaling pathways, and these changes are mediated by FoxO transcription factors.
    Keywords:  FoxO transcription factors; RNA sequencing; calcium signaling; diabetes; insulin/IGF-1 receptors; mitochondrial dysfunction; muscle transcription
  8. Cells. 2022 Feb 17. pii: 702. [Epub ahead of print]11(4):
      The mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol-3 kinase (PI3K)/AKT pathways are dysregulated in various human cancers, including pancreatic ductal adenocarcinoma (PDAC), which has a very poor prognosis due to its lack of efficient therapies. We have previously identified ACAGT-007a (GT-7), an anti-cancer compound that kills ERK-active melanoma cells by inducing ERK-dependent apoptosis. Here, we investigated the apoptosis-inducing effect of GT-7 on three PDAC cell lines and its relevance with the MAPK/ERK and PI3K/AKT signaling pathways. GT-7 induced apoptosis in PDAC cells with different KRAS mutations (MIA-Pa-Ca-2 (KRAS G12C), T3M4 (KRAS Q61H), and PANC-1 (KRAS G12D)), being T3M4 most susceptible, followed by MIA-Pa-Ca-2, and PANC-1 was most resistant to apoptosis induction by GT-7. GT-7 stimulated ERK phosphorylation in the three PDAC cells, but only T3M4 displayed ERK-activation-dependent apoptosis. Furthermore, GT-7 induced a marked down-regulation of AKT phosphorylation after a transient peak in T3M4, whereas PANC-1 displayed the strongest and most sustained AKT activation, followed by MIA-Pa-Ca-2, suggesting that sustained AKT phosphorylation as a determinant for the resistance to GT-7-mediated apoptosis. Consistently, a PI3K inhibitor, Wortmannin, abolished AKT phosphorylation and enhanced GT-7-mediated apoptosis in T3M4 and MIA-Pa-Ca-2, but not in PANC-1, which showed residual AKT phosphorylation. This is the first report that ERK stimulation alone or in combination with AKT signaling inhibition can effectively induce apoptosis in PDAC and provides a rationale for a novel concurrent targeting of the PI3K/AKT and ERK pathways.
    Keywords:  ACAGT-007a (GT-7); AKT signaling; ERK MAPK; KRAS; apoptosis; pancreatic cancer
  9. Elife. 2022 02 24. pii: e66869. [Epub ahead of print]11
      Kinase activity in signaling networks frequently depends on regulatory subunits that can both inhibit activity by interacting with the catalytic subunits and target the kinase to distinct molecular partners and subcellular compartments. Here, using a new synthetic molecular interaction system, we show that translocation of a regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane has a paradoxical effect on the membrane kinase activity. It can both enhance it at lower translocation levels, even in the absence of signaling inputs, and inhibit it at higher translocation levels, suggesting its role as a linker that can both couple and decouple signaling processes in a concentration-dependent manner. We further demonstrate that superposition of gradients of PKA-R abundance across single cells can control the directionality of cell migration, reversing it at high enough input levels. Thus, complex in vivo patterns of PKA-R localization can drive complex phenotypes, including cell migration.
    Keywords:  FRET; PKA; biochemistry; cell migration; chemical biology; kinase activity; microfluidics; none; physics of living systems; signal transduction
  10. J Biol Chem. 2022 Feb 17. pii: S0021-9258(22)00184-3. [Epub ahead of print] 101744
      The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by ten-fold in the Ragulator-deficient clone compared to that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.
    Keywords:  Ragulator; lysosome; mammalian target of rapamycin (mTOR); nuclear translocation; scaffold protein; transcription factor EB
  11. Cancers (Basel). 2022 Feb 09. pii: 860. [Epub ahead of print]14(4):
      The proliferation and survival signals emanating from the B-cell receptor (BCR) constitute a crucial aspect of mature lymphocyte's life. Dysregulated BCR signaling is considered a potent contributor to tumor survival in different subtypes of B-cell non-Hodgkin lymphomas (B-NHLs). In the last decade, the emergence of BCR-associated kinases as rational therapeutic targets has led to the development and approval of several small molecule inhibitors targeting either Bruton's tyrosine kinase (BTK), spleen tyrosine kinase (SYK), or phosphatidylinositol 3 kinase (PI3K), offering alternative treatment options to standard chemoimmunotherapy, and making some of these drugs valuable assets in the anti-lymphoma armamentarium. Despite their initial effectiveness, these precision medicine strategies are limited by primary resistance in aggressive B-cell lymphoma such as diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), especially in the case of first generation BTK inhibitors. In these patients, BCR-targeting drugs often fail to produce durable responses, and nearly all cases eventually progress with a dismal outcome, due to secondary resistance. This review will discuss our current understanding of the role of antigen-dependent and antigen-independent BCR signaling in DLBCL and MCL and will cover both approved inhibitors and investigational molecules being evaluated in early preclinical studies. We will discuss how the mechanisms of action of these molecules, and their off/on-target effects can influence their effectiveness and lead to toxicity, and how our actual knowledge supports the development of more specific inhibitors and new, rationally based, combination therapies, for the management of MCL and DLBCL patients.
    Keywords:  B-cell non-Hodgkin lymphoma (B-NHL); B-cell receptor (BCR); Bruton’s tyrosine kinase (BTK); acalabrutinib; combination therapies; ibrutinib; phosphoinositide-3-kinase (PI3K); spleen tyrosine kinase (SYK)
  12. FASEB J. 2022 03;36(3): e22211
      Metabolic dysfunction and insulin resistance are emerging as hallmarks of cancer and cachexia, and impair cancer prognosis. Yet, the molecular mechanisms underlying impaired metabolic regulation are not fully understood. To elucidate the mechanisms behind cancer-induced insulin resistance in muscle, we isolated extensor digitorum longus (EDL) and soleus muscles from Lewis Lung Carcinoma tumor-bearing mice. Three weeks after tumor inoculation, muscles were isolated and stimulated with or without a submaximal dose of insulin (1.5 nM). Glucose transport was measured using 2-[3 H]Deoxy-Glucose and intramyocellular signaling was investigated using immunoblotting. In soleus muscles from tumor-bearing mice, insulin-stimulated glucose transport was abrogated concomitantly with abolished insulin-induced TBC1D4 and GSK3 phosphorylation. In EDL, glucose transport and TBC1D4 phosphorylation were not impaired in muscles from tumor-bearing mice, while AMPK signaling was elevated. Anabolic insulin signaling via phosphorylation of the mTORC1 targets, p70S6K thr389, and ribosomal-S6 ser235, were decreased by cancer in soleus muscle while increased or unaffected in EDL. In contrast, the mTOR substrate, pULK1 ser757, was reduced in both soleus and EDL by cancer. Hence, cancer causes considerable changes in skeletal muscle insulin signaling that is dependent on muscle-type, which could contribute to metabolic dysregulation in cancer. Thus, the skeletal muscle could be a target for managing metabolic dysfunction in cancer.
    Keywords:  AMPK; Akt; Lewis lung carcinoma; TBC1D4; cachexia; cancer; glucose metabolism; insulin resistance; mTORC1; muscle
  13. Trends Cancer. 2022 Feb 17. pii: S2405-8033(22)00021-8. [Epub ahead of print]
      Epithelial-mesenchymal plasticity (EMP) reflects the capacity of cells to interconvert between epithelial and mesenchymal phenotypes. In cancer, these dynamics ultimately contribute to disease progression. Despite decades of study, a consistent molecular definition of this plasticity remains elusive because of its inherent variability. The advent of quantitative single-cell biology is unveiling unexpected complexity, and new conceptual frameworks are required to understand the emergence and relevance of EMP in cancer. Here, we use principles from multitask optimization to propose that EMP reflects an adaptive response of epithelial cells in response to homeostatic disruption, giving rise to generalist phenotypes. We use this theory to predict properties of these cells and their contribution to tumor progression.
    Keywords:  EMT; Pareto optimality; adaptation; epithelial-mesenchymal plasticity; evolution; plasticity
  14. Life (Basel). 2022 Feb 14. pii: 283. [Epub ahead of print]12(2):
      Chronic lymphocytic leukemia (CLL), the most common type of leukemia in adults, is characterized by a high degree of clinical heterogeneity that is influenced by the disease's molecular complexity. The genes most frequently affected in CLL cluster into specific biological pathways, including B-cell receptor (BCR) signaling, apoptosis, NF-κB, and NOTCH1 signaling. BCR signaling and the apoptosis pathway have been exploited to design targeted medicines for CLL therapy. Consistently, molecules that selectively inhibit specific BCR components, namely Bruton tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) as well as inhibitors of BCL2, have revolutionized the therapeutic management of CLL patients. Several BTK inhibitors and PI3K inhibitors with different modes of action are currently used or are in development in advanced stage clinical trials. Moreover, the restoration of apoptosis by the BCL2 inhibitor venetoclax offers meaningful clinical activity with a fixed-duration scheme. Inhibitors of the BCR and of BCL2 are able to overcome the chemorefractoriness associated with high-risk genetic features, including TP53 disruption. Other signaling cascades involved in CLL pathogenesis, in particular NOTCH signaling and NF-kB signaling, already provide biomarkers for a precision medicine approach to CLL and may represent potential druggable targets for the future. The aim of the present review is to discuss the druggable pathways of CLL and to provide the biological background of the high efficacy of targeted biological drugs in CLL.
    Keywords:  chronic lymphocytic leukemia; precision medicine; target therapy
  15. Nature. 2022 Feb 23.
      Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.
  16. Allergy Asthma Clin Immunol. 2022 Feb 21. 18(1): 15
      BACKGROUND: Activated phosphoinositide 3-kinase (PI3K) δ syndrome (APDS) is a rare form of primary immunodeficiency with 243 known cases reported in the literature. Known findings associated with the condition include recurrent sinusitis and bronchitis, bronchiectasis, immune cytopenias, mild developmental delay, splenomegaly, and lymphadenopathy. We report the case of a child with APDS accompanied by unique clinical features: nephromegaly and growth hormone deficiency with associated pituitary anatomic abnormality.CASE PRESENTATION: The patient is a nine-year-old boy with a heterozygous de novo variant in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit δ (p.E1021K), previously reported in association with APDS. Our patient, who had no family history of immunodeficiency, exhibits classic findings of this syndrome but also has unique features that extend the phenotypic spectrum of this disorder. At 5 years of age, the patient showed marked growth deceleration and was demonstrated to have growth hormone (GH) deficiency with associated pituitary anatomic abnormality. He started GH therapy with an excellent response. He additionally has bilateral nephromegaly of unclear etiology, microscopic hematuria and proteinuria, asthma, and has developed left hip pain with arthrocentesis consistent with oligoarticular juvenile idiopathic arthritis. At age nine, the patient was referred to genetics and whole exome sequencing revealed APDS. Though there was initial concern that GH may increase risk for malignancy as GH signals through the PI3K pathway, he was allowed to continue treatment as the PI3K pathway was considered constitutively active at baseline.
    CONCLUSIONS: Our patient's unique presentation adds to the clinical information regarding APDS, demonstrates the utility of genetic testing and illustrates the importance of a multidisciplinary collaborative approach in managing this complex syndrome.
    Keywords:  Asthma; Bronchiectasis; Growth hormone deficiency; Nephromegaly; Pituitary abnormality; Primary immunodeficiency
  17. J Biol Chem. 2022 Feb 16. pii: S0021-9258(22)00180-6. [Epub ahead of print] 101740
      Lysosomes serve as dynamic regulators of cell and organismal physiology by integrating the degradation of macromolecules with receptor and nutrient signaling. Previous studies have established that activation of the transcription factors TFEB and TFE3 induces the expression of lysosomal genes and proteins in signaling-inactive starved cells, that is, under conditions when activity of the master regulator of nutrient-sensing signaling mTORC1 is repressed. How lysosome biogenesis is triggered in signaling-active cells is incompletely understood. Here we identify a role for calcium release from the lumen of the endoplasmic reticulum (ER) in the control of lysosome biogenesis that is independent of mTORC1. We show using functional imaging that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon depletion of INPP5A, an inositol 5-phosphatase downregulated in cancer and defective in spinocerebellar ataxia, or receptor-mediated phospholipase C activation leads to the induction of lysosome biogenesis. This mechanism involves calcineurin and the nuclear translocation and elevated transcriptional activity of TFEB/ TFE3. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lysosome biogenesis via TFEB/ TFE3, thereby contributing to our understanding how cells are able to maintain their lysosome content under conditions of active receptor and nutrient signaling.
    Keywords:  calcium; imaging; inositol-triphosphate; lysosome biogenesis; signaling
  18. Mol Cell Pediatr. 2022 Feb 21. 9(1): 3
      BACKGROUND: The PTEN hamartoma tumor syndrome (PHTS) encompasses several different syndromes, which are linked to an autosomal-dominant mutation of the tumor suppressor PTEN gene on chromosome 10. Loss of PTEN activity leads to an increased phosphorylation of different cell proteins, which may have an influence on growth, migration, and apoptosis. Excessive activity of the PI3K/AKT/mTOR pathway due to PTEN deficiency may lead to the development of benign and malignant tumors and overgrowth. Diagnosis of PHTS in childhood can be even more challenging than in adulthood because of a lack of well-defined diagnostic criteria. So far, there are no official recommendations for cancer surveillance in affected children and adolescents.MAIN BODY: All individuals with PHTS are at high risk for tumor development and thus might benefit from cancer surveillance strategies. In childhood, macrocephaly may be the only evident symptom, but developmental delay, behavioral problems, dermatological features (e.g., penile freckling), vascular anomalies, lipoma, or enlarged perivascular spaces in cerebral magnetic resonance imaging (cMRI) may help to establish the diagnosis. Regular psychomotor assessment and assistance in subjects with neurological impairment play an important role in the management of affected children. Already in early childhood, affected patients bear a high risk to develop thyroid pathologies. For that reason, monitoring of thyroid morphology and function should be established right after diagnosis. We present a detailed description of affected organ systems, tools for initiation of molecular diagnostic and screening recommendations for patients < 18 years of age.
    CONCLUSION: Affected families frequently experience a long way until the correct diagnosis for their child's peculiarity is made. Even after diagnosis, it is not easy to find a physician who is familiar with this rare group of diseases. Because of a still-limited database, it is not easy to establish evidence-based (cancer) surveillance recommendations. The presented screening recommendation should thus be revised regularly according to the current state of knowledge.
    Keywords:  Adolescence; Cancer surveillance; Childhood; Diagnostic; Guideline; Management; PHTS; Treatment
  19. Proc Natl Acad Sci U S A. 2022 Mar 01. pii: e2103532119. [Epub ahead of print]119(9):
      An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth.
    Keywords:  CRISPR; cancer; double-strand breaks; insertion/deletion
  20. Nat Rev Cancer. 2022 Feb 22.
      Over the past decade, CRISPR has become as much a verb as it is an acronym, transforming biomedical research and providing entirely new approaches for dissecting all facets of cell biology. In cancer research, CRISPR and related tools have offered a window into previously intractable problems in our understanding of cancer genetics, the noncoding genome and tumour heterogeneity, and provided new insights into therapeutic vulnerabilities. Here, we review the progress made in the development of CRISPR systems as a tool to study cancer, and the emerging adaptation of these technologies to improve diagnosis and treatment.
  21. Dev Cell. 2022 Feb 15. pii: S1534-5807(22)00069-7. [Epub ahead of print]
      Human pluripotent stem cells (hPSCs) can self-renew indefinitely or can be induced to differentiate. We previously showed that exogenous glutamine (Gln) withdrawal biased hPSC differentiation toward ectoderm and away from mesoderm. We revealed that, although all three germ lineages are capable of de novo Gln synthesis, only ectoderm generates sufficient Gln to sustain cell viability and differentiation, and this finding clarifies lineage fate restrictions under Gln withdrawal. Furthermore, we found that Gln acts as a signaling molecule for ectoderm that supersedes lineage-specifying cytokine induction. In contrast, Gln in mesoderm and endoderm is the preferred precursor of α-ketoglutarate without a direct signaling role. Our work raises a question about whether the nutrient environment functions directly in cell differentiation during development. Interestingly, transcriptome analysis of a gastrulation-stage human embryo shows that unique Gln enzyme-encoding gene expression patterns may also distinguish germ lineages in vivo. Together, our study suggests that intracellular Gln may help coordinate differentiation of the three germ layers.
    Keywords:  auxotroph; cell fate; development; glutamine; nutrient; pluripotent stem cell; prototroph
  22. Cancers (Basel). 2022 Feb 20. pii: 1067. [Epub ahead of print]14(4):
      The dysregulation of PI3K, HDACs, and MYCN are well known for promoting multiple cancer types, including neuroblastoma (NB). Targeting the upstream regulators of MYCN, including HDACs and PI3K, was shown to suppress cancer growth. In the present study, we analyze different NB patient datasets to reveal that high PI3K and HDAC expression is correlated with overall poor NB patient survival. High PI3K level is also found to be associated with high MYCN level and NB stage progression. We repurpose a dual inhibitor CUDC-907 as a single agent to directly target both PI3K and HDAC in NB. We use in vitro methodologies to determine the efficacy and selectivity of CUDC-907 using six NB and three control fibroblast cell lines. Our results show that CUDC-907 significantly inhibits NB proliferation and colony growth, induces apoptosis, blocks cell cycle progression, inhibits MYCN, and enhances H3K9Ac levels by inhibiting the PI3K/AKT signaling pathway and HDAC function. Furthermore, CUDC-907 significantly inhibits NB tumor growth in a 3D spheroid tumor model that recapitulates the in vivo tumor growth. Overall, our findings highlight that the dual inhibition of PI3K and HDAC by CUDC-907 is an effective therapeutic strategy for NB and other MYC-dependent cancers.
    Keywords:  Fimepinostat; PI3K pathway; drug repurposing; epigenetics; neuroblastoma; pediatric cancer
  23. Front Immunol. 2022 ;13 797244
      PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαβ+ T-ALL development. Herein, we explored the functional interaction between TCRαβ signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Ptendel blasts were unable to release calcium ions (Ca2+) from the endoplasmic reticulum to the cytosol. In order to understand such phenomena, we constructed a mathematical model centered on the mechanisms controlling the calcium flux, integrating TCR signal strength and PTEN interactions. This qualitative model displays a dynamical behavior coherent with the dynamics reported in the literature, it also predicts that PTEN affects positively IP3 (inositol 1,4,5-trisphosphate) receptors (ITPR). Hence, we analyzed Itpr expression and unraveled that ITPR proteins levels are reduced in PTEN-deficient tumor cells compared to physiological and leukemic PTEN-proficient cells. However, calcium flux and ITPR proteins expression are not defective in non-leukemic PTEN-deficient T cells indicating that beyond PTEN loss an additional alteration is required. Altogether, our study shows that ITPR/Calcium flux is a part of the oncogenic landscape shaped by PTEN loss and pinpoints a putative role of PTEN in the regulation of ITPR proteins in thymocytes, which remains to be characterized.
    Keywords:  PTEN; T-ALL; TCR signaling; calcium signaling; qualitative mathematical model; single-cell RNA-seq; thymocytes
  24. Genes (Basel). 2022 Jan 31. pii: 287. [Epub ahead of print]13(2):
      Cervical cancer is the fourth leading cause of cancer-related deaths in women worldwide. Although many sequencing studies have been carried out, the genetic characteristics of cervical cancer remain to be fully elucidated, especially in the Asian population. Herein, we investigated the genetic landscape of Chinese cervical cancer patients using a validated multigene next generation sequencing (NGS) panel. We analyzed 64 samples, consisting of 32 tumors and 32 blood samples from 32 Chinese cervical cancer patients by performing multigene NGS with a panel targeting 571 cancer-related genes. A total of 810 somatic variants, 2730 germline mutations and 701 copy number variations (CNVs) were identified. FAT1, HLA-B, PIK3CA, MTOR, KMT2D and ZFHX3 were the most mutated genes. Further, PIK3CA, BRCA1, BRCA2, ATM and TP53 gene loci had a higher frequency of CNVs. Moreover, the role of PIK3CA in cervical cancer was further highlighted by comparing with the ONCOKB database, especially for E545K and E542K, which were reported to confer radioresistance to cervical cancer. Notably, analysis of potential therapeutic targets suggested that cervical cancer patients could benefit from PARP inhibitors. This multigene NGS analysis revealed several novel genetic alterations in Chinese patients with cervical cancer and highlighted the role of PIK3CA in cervical cancer. Overall, this study showed that genetic variations not only affect the genetic susceptibility of cervical cancer, but also influence the resistance of cervical cancer to radiotherapy, but further studies involving a larger patient population should be undertaken to validate these findings.
    Keywords:  PIK3CA; cervical cancer; genetic traits; multigene NGS panel; therapeutic target
  25. J Vis Exp. 2022 Feb 03.
      Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate. Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers, DNA content and type. The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.
  26. N Engl J Med. 2022 02 24. 386(8): 735-743
      BACKGROUND: Covalent (irreversible) Bruton's tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood.METHODS: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors.
    RESULTS: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell-receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors.
    CONCLUSIONS: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.).
  27. BMC Genomics. 2022 Feb 22. 23(1): 156
      BACKGROUND: Patient-derived xenografts (PDX) mice models play an important role in preclinical trials and personalized medicine. Sharing data on the models is highly valuable for numerous reasons - ethical, economical, research cross validation etc. The EurOPDX Consortium was established 8 years ago to share such information and avoid duplicating efforts in developing new PDX mice models and unify approaches to support preclinical research. EurOPDX Data Portal is the unified data sharing platform adopted by the Consortium.MAIN BODY: In this paper we describe the main features of the EurOPDX Data Portal ( ), its architecture and possible utilization by researchers who look for PDX mice models for their research. The Portal offers a catalogue of European models accessible on a cooperative basis. The models are searchable by metadata, and a detailed view provides molecular profiles (gene expression, mutation, copy number alteration) and treatment studies. The Portal displays the data in multiple tools (PDX Finder, cBioPortal, and GenomeCruzer in future), which are populated from a common database displaying strictly mutually consistent views.
    (SHORT) CONCLUSION: EurOPDX Data Portal is an entry point to the EurOPDX Research Infrastructure offering PDX mice models for collaborative research, (meta)data describing their features and deep molecular data analysis according to users' interests.
    Keywords:  Data harmonization; Database; Molecular data analysis; PDX; Research infrastructure