Impact of therapeutic inhibition of oncogenic cell signaling tyrosine kinase on cell metabolism: in vivo-detectable metabolic biomarkers of inhibition.

BACKGROUND: Inhibition of kinases is the ever-expanding therapeutic approach to various types of cancer. Typically, assessment of the treatment response is accomplished by standard, volumetric imaging procedures, performed weeks to months after the onset of treatment, given the predominantly cytostatic nature of the kinase inhibitors, at least when used as single agents. Therefore, there is a great clinical need to develop new monitoring approaches to detect the response to kinase inhibition much more promptly. Noninvasive 1H magnetic resonance spectroscopy (MRS) can measure in vitro and in vivo concentration of key metabolites which may potentially serve as biomarkers of response to kinase inhibition. METHODS: We employed mantle cell lymphoma (MCL) cell lines demonstrating markedly diverse sensitivity of inhibition of Bruton's tyrosine kinase (BTK) regarding their growth and studied in-depth effects of the inhibition on various aspects of cell metabolism including metabolite synthesis using metabolomics, glucose and oxidative metabolism by Seahorse XF technology, and concentration of index metabolites lactate, alanine, total choline and taurine by 1H MRS. RESULTS: Effective BTK inhibition profoundly suppressed key cell metabolic pathways, foremost pyrimidine and purine synthesis, the citrate (TCA) cycle, glycolysis, and pyruvate and glutamine/alanine metabolism. It also inhibited glycolysis and amino acid-related oxidative metabolism. Finally, it profoundly and quickly decreased concentration of lactate (a product of mainly glycolysis) and alanine (an indicator of amino acid metabolism) and, less universally total choline both in vitro and in vivo, in the MCL xenotransplant model. The decrease correlated directly with the degree of inhibition of lymphoma cell expansion and tumor growth. CONCLUSIONS: Our results indicate that BTK inhibition exerts a broad and profound suppressive effect on cell metabolism and that the affected index metabolites such as lactate, alanine may serve as early, sensitive, and reliable biomarkers of inhibition in lymphoma patients detectable by noninvasive MRS-based imaging method. This kind of imaging-based detection may also be applicable to other kinase inhibitors, as well as diverse lymphoid and non-lymphoid malignancies.

Induction of Transcriptional Inhibitor HES1 and the Related Repression of Tumor-Suppressor TXNIP Are Important Components of Cell-Transformation Program Imposed by Oncogenic Kinase NPM-ALK.

This study reports that hairy and enhancer of split homolog-1 (HES1), known to repress gene transcription in progenitor cells of several cell lineages, was strongly expressed in cells and tissues of T-cell lymphoma expressing the oncogenic chimeric tyrosine kinase nucleophosmin (NPM)-anaplastic lymphoma kinase [ALK; ALK+ T-cell lymphoma (TCL)]. The structural analysis of the Orange domain of HES1 indicated that HES1 formed a highly stable homodimer. Of note, repression of HES1 expression led to inhibition of ALK+ TCL cell growth in vivo. The expression of the HES1 gene was induced by NPM-ALK through activation of STAT3, which bound to the gene's promoter and induced the gene's transcription. NPM-ALK also directly phosphorylated HES1 protein. In turn, HES1 up-regulated and down-regulated in ALK+ TCL cells, the expression of numerous genes, protein products of which are involved in key cell functions, such as cell proliferation and viability. Among the genes inhibited by HES1 was thioredoxin-interacting protein (TXNIP), encoding a protein implicated in promotion of cell death in various types of cells. Accordingly, ALK+ TCL cells and tissues lacked expression of TXNIP, and its transcription was co-inhibited by HES1 and STAT3 in an NPM-ALK-dependent manner. Finally, the induced expression of TXNIP induced massive apoptotic cell death of ALK+ TCL cells. The results reveal a novel NPM-ALK-controlled pro-oncogenic regulatory network and document an important role of HES and TXNIP in the NPM-ALK-driven oncogenesis, with the former protein displaying oncogenic and the latter tumor suppressor properties.

Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control.

Ubiquitinylation drives many cellular processes by targeting proteins for proteasomal degradation. Ubiquitin conjugation enzymes promote ubiquitinylation and, thus, degradation of protein substrates. Ubiquitinylation is a well-known posttranslational modification controlling cell-cycle transitions and levels or/and activation levels of ubiquitin-conjugating enzymes change during development and cell cycle. Progression through the cell cycle is tightly controlled by CDK inhibitors such as p27Kip1. Here we show that, in contrast to promoting its degradation, the ubiquitin-conjugating enzyme UBCH7/UBE2L3 specifically protects p27Kip1 from degradation. Overexpression of UBCH7/UBE2L3 stabilizes p27Kip1 and delays the G1-to-S transition, while depletion of UBCH7/UBE2L3 increases turnover of p27Kip1. Levels of p21Cip1/Waf1, p57Kip2, cyclin A and cyclin E, all of which are also involved in regulating the G1/S transition are not affected by UBCH7/UBE2L3 depletion. The effect of UBCH7/UBE2L3 on p27Kip1 is not due to alteration of the levels of any of the ubiquitin ligases known to ubiquitinylate p27Kip1. Rather, UBCH7/UBE2L3 catalyzes the conjugation of heterotypic ubiquitin chains on p27Kip1 that are proteolytically incompetent. These data reveal new controls and concepts about the ubiquitin proteasome system in which a ubiquitin-conjugating enzyme selectively inhibits and may even protect, rather than promote degradation of a crucial cell-cycle regulatory molecule.-Whitcomb, E. A., Tsai, Y. C., Basappa, J., Liu, K., Le Feuvre, A. K., Weissman, A. M., Taylor, A. Stabilization of p27Kip1/CDKN1B by UBCH7/UBE2L3 catalyzed ubiquitinylation: a new paradigm in cell-cycle control.

Pyrimidine tract-binding protein 1 mediates pyruvate kinase M2-dependent phosphorylation of signal transducer and activator of transcription 3 and oncogenesis in anaplastic large cell lymphoma.

PKM2 (pyruvate kinase M2), a critical regulator of glycolysis, is phosphorylated by numerous growth factor receptors and oncogenic tyrosine kinases including NPM-ALK which is expressed in a subset of aggressive T-cell non-Hodgkin lymphomas known as anaplastic large cell lymphoma, ALK-positive. Our previous work demonstrated that phosphorylation of Y105-PKM2 by NPM-ALK regulates a major metabolic shift to promote lymphomagenesis. In addition to its role in metabolism, recent studies have shown that PKM2 promotes oncogenesis by phosphorylating nuclear STAT3 (signal transducer and activator of transcription 3) and regulating transcription of genes involved in cell survival and proliferation. We hypothesized that identification of novel PKM2 interactors could provide additional insights into its expanding functional role in cancer. To this end, immunocomplexes of FLAG-tagged PKM2 were isolated from NPM-ALK-positive ALCL (anaplastic large cell lymphoma) cells and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) which led to the identification of polypyrimidine tract-binding protein (PTBP1) as a novel interactor of PKM2. The interaction between PTBP1 and PKM2 was restricted to the nucleus and was dependent on NPM-ALK mediated Y105 phosphorylation of PKM2. Stable shRNA-mediated silencing of PTBP1 resulted in a marked decrease in pY105-PKM2 and pY705-STAT3 which led to decreased ALCL cell proliferation and colony formation. Overall, our data demonstrate that PTBP1 interacts with PKM2 and promotes ALCL oncogenesis by facilitating PKM2-dependent activation of STAT3 within the nucleus.

Chimeric kinase ALK induces expression of NAMPT and selectively depends on this metabolic enzyme to sustain its own oncogenic function.

As we show in this study, NAMPT, the key rate-limiting enzyme in the salvage pathway, one of the three known pathways involved in NAD synthesis, is selectively over-expressed in anaplastic T-cell lymphoma carrying oncogenic kinase NPM1::ALK (ALK + ALCL). NPM1::ALK induces expression of the NAMPT-encoding gene with STAT3 acting as transcriptional activator of the gene. Inhibition of NAMPT affects ALK + ALCL cells expression of numerous genes, many from the cell-signaling, metabolic, and apoptotic pathways. NAMPT inhibition also functionally impairs the key metabolic and signaling pathways, strikingly including enzymatic activity and, hence, oncogenic function of NPM1::ALK itself. Consequently, NAMPT inhibition induces cell death in vitro and suppresses ALK + ALCL tumor growth in vivo. These results indicate that NAMPT is a novel therapeutic target in ALK + ALCL and, possibly, other similar malignancies. Targeting metabolic pathways selectively activated by oncogenic kinases to which malignant cells become "addicted" may become a novel therapeutic approach to cancer, alternative or, more likely, complementary to direct inhibition of the kinase enzymatic domain. This potential therapy to simultaneously inhibit and metabolically "starve" oncogenic kinases may not only lead to higher response rates but also delay, or even prevent, development of drug resistance, frequently seen when kinase inhibitors are used as single agents.

A corticotropin-releasing factor system expressed in the cochlea modulates hearing sensitivity and protects against noise-induced hearing loss.

Noise-induced hearing loss is a highly prevalent occupational injury, yet little is known concerning the signals controlling normal cochlear sensitivity and susceptibility to noise-induced trauma. While the corticotropin-releasing factor (CRF) system is involved in activation of the classic hypothalamic-pituitary-adrenal axis, it is also involved in local physiological responses to stress in many tissues, and is expressed in the inner ear. We demonstrate that mice lacking the CRF receptor CRFR2 exhibit a significantly lower auditory threshold than wild type mice, but this gain of function comes at the price of increased susceptibility to acoustic trauma. We further demonstrate that glutamatergic transmission, purinergic signaling, and activation of Akt (PKB) pathways within the cochlea are misregulated, which may underlie the enhanced sensitivity and trauma susceptibility observed in CRFR2(-/-) mice. Our data suggest that CRFR2 constitutively modulates hearing sensitivity under normal conditions, and thereby provides protection against noise-induced hearing loss.

Corticotropin-releasing factor-2 activation prevents gentamicin-induced oxidative stress in cells derived from the inner ear.

Generation of reactive oxygen species (ROS) is a common denominator in many conditions leading to cell death in the cochlea, yet little is known of the cochlea's endogenous mechanisms involved in preventing oxidative stress and its consequences in the cochlea. We have recently described a corticotropin-releasing factor (CRF) signaling system in the inner ear involved in susceptibility to noise-induced hearing loss. We use biochemical and proteomics assays to define further the role of CRF signaling in the response of cochlear cells to aminoglycoside exposure. We demonstrate that activity via the CRF(2) class of receptors protects against aminoglycoside-induced ROS production and activation of cell death pathways. This study suggests for the first time a role for CRF signaling in protecting the cochlea against oxidative stress, and our proteomics data suggest novel mechanisms beyond induction of free radical scavengers that are involved in its protective mechanisms.

ACLY is the novel signaling target of PIP2/PIP3 and Lyn in acute myeloid leukemia.

A fundamental feature of tumor progression is reprogramming of metabolic pathways. ATP citrate lyase (ACLY) is a key metabolic enzyme that catalyzes the generation of Acetyl-CoA and is upregulated in cancer cells and required for their growth. The phosphoinositide 3-kinase (PI3K) and Src-family kinase (SFK) Lyn are constitutively activate in many cancers. We show here, for the first time, that both the substrate and product of PI3K, phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), respectively, bind to ACLY in Acute Myeloid Leukemia (AML) patient-derived, but not normal donor-derived cells. We demonstrate the binding of PIP2 to the CoA-binding domain of ACLY and identify the six tyrosine residues of ACLY that are phosphorylated by Lyn. Three of them (Y682, Y252, Y227) can be also phosphorylated by Src and they are located in catalytic, citrate binding and ATP binding domains, respectively. PI3K and Lyn inhibitors reduce the ACLY enzyme activity, ACLY-mediated Acetyl-CoA synthesis, phospholipid synthesis, histone acetylation and cell growth. Thus, PIP2/PIP3 binding and Src tyrosine kinases-mediated stimulation of ACLY links oncogenic pathways to Acetyl-CoA-dependent pro-growth and survival metabolic pathways in cancer cells. These results indicate a novel function for Lyn, as a regulator of Acetyl-CoA-mediated metabolic pathways.

Multiplexed isobaric tagging protocols for quantitative mass spectrometry approaches to auditory research.

Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a complex lysate for the ultimate differential quantification of protein abundance following experimental manipulations. Following a successful outcome of the labeling procedure, the sample is submitted for mass spectrometric analysis, resulting in peptide quantification and protein identification. While we will concentrate on cells in culture, we will point out procedures that can be used for labeling lysates generated from other tissues, along with any minor modifications required for such samples. We will also outline, but not fully document, other strategies used in our lab to label proteins prior to mass spectrometric analysis, and describe under which conditions each procedure may be desirable. What is not covered in this chapter is anything but the most brief introduction to mass spectrometry (instrumentation, theory, etc.), nor do we attempt to cover much in the way of software used for post hoc analysis. These two topics are dependant upon one's resources, and where applicable, one's collaborators. We strongly encourage the reader to seek out expert advice on topics not covered here.

Multiplexed Isobaric Tagging Protocols for Quantitative Mass Spectrometry Approaches to Auditory Research.

Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a complex lysate for the ultimate differential quantification of protein abundance following experimental manipulations. Following a successful outcome of the labeling procedure, the sample is submitted for mass spectrometric analysis, resulting in peptide quantification and protein identification. While we will concentrate on cells in culture, we will point out procedures that can be used for labeling lysates generated from tissues, along with any minor modifications required for such samples. We will also outline, but not fully document, other strategies used in our lab to label proteins prior to mass spectrometric analysis, and describe under which conditions each procedure may be desirable. What is not covered in this chapter is anything but the most brief introduction to mass spectrometry (instrumentation, theory, etc.), nor do we attempt to cover much in the way of software used for post hoc analysis. These two topics are dependent upon one's resources, and where applicable, one's collaborators. We strongly encourage the reader to seek out expert advice on topics not covered here.

The cochlea as an independent neuroendocrine organ: expression and possible roles of a local hypothalamic-pituitary-adrenal axis-equivalent signaling system.

A key property possessed by the mammalian cochlea is its ability to dynamically alter its own sensitivity. Because hair cells and ganglion cells are prone to damage following exposure to loud sound, extant mechanisms limiting cochlear damage include modulation involving both the mechanical (via outer hair cell motility) and neural signaling (via inner hair cell-ganglion cell synapses) steps of peripheral auditory processing. Feedback systems such as that embodied by the olivocochlear system can alter sensitivity, but respond only after stimulus encoding, allowing potentially damaging sounds to impact the inner ear before sensitivity is adjusted. Less well characterized are potential cellular signaling systems involved in protection against metabolic stress and resultant damage. Although pharmacological manipulation of the olivocochlear system may hold some promise for attenuating cochlear damage, targeting this system may still allow damage to occur that does not depend on a fully functional feedback loop for its mitigation. Thus, understanding endogenous cell signaling systems involved in cochlear protection may lead to new strategies and therapies for prevention of cochlear damage and consequent hearing loss. We have recently discovered a novel cochlear signaling system that is molecularly equivalent to the classic hypothalamic-pituitary-adrenal (HPA) axis. This cochlear HPA-equivalent system functions to balance auditory sensitivity and susceptibility to noise-induced hearing loss, and also protects against cellular metabolic insults resulting from exposures to ototoxic drugs. This system may represent a local cellular response system designed to mitigate damage arising from various types of insult.

The cochlear CRF signaling systems and their mechanisms of action in modulating cochlear sensitivity and protection against trauma.

A key requirement for encoding the auditory environment is the ability to dynamically alter cochlear sensitivity. However, merely attaining a steady state of maximal sensitivity is not a viable solution since the sensory cells and ganglion cells of the cochlea are prone to damage following exposure to loud sound. Most often, such damage is via initial metabolic insult that can lead to cellular death. Thus, establishing the highest sensitivity must be balanced with protection against cellular metabolic damage that can lead to loss of hair cells and ganglion cells, resulting in loss of frequency representation. While feedback mechanisms are known to exist in the cochlea that alter sensitivity, they respond only after stimulus encoding, allowing potentially damaging sounds to impact the inner ear at times coincident with increased sensitivity. Thus, questions remain concerning the endogenous signaling systems involved in dynamic modulation of cochlear sensitivity and protection against metabolic stress. Understanding endogenous signaling systems involved in cochlear protection may lead to new strategies and therapies for prevention of cochlear damage and consequent hearing loss. We have recently discovered a novel cochlear signaling system that is molecularly equivalent to the classic hypothalamic-pituitary-adrenal (HPA) axis. This cochlear HPA-equivalent system functions to balance auditory sensitivity and susceptibility to noise-induced hearing loss, and also protects against cellular metabolic insults resulting from exposures to ototoxic drugs. We review the anatomy, physiology, and cellular signaling of this system, and compare it to similar signaling in other organs/tissues of the body.