Johannes Roesel

Tyrosine phosphorylation inhibits PKM2 to promote the Warburg effect and tumor growth

Tyrosine phosphorylation of pyruvate kinase M2 gives tumor cells a metabolic advantage. A Malignant Metabolic Switch Cancer cells show aberrant metabolism, consuming more glucose than do healthy cells and producing lactate even in the presence of abundant oxygen, rather than shifting to oxidative phosphorylation. This phenomenon is called the Warburg effect, after Otto Warburg, who described it many years ago. Building on recent research implicating inhibition of the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2) by phosphotyrosine binding as critical to the Warburg effect—and tumorigenesis—Hitosugi et al. explored the role of signaling from oncogenic forms of the fibroblast growth factor receptor type 1 (FGFR1) in mediating this metabolic switch. They found that FGFR1, a receptor tyrosine kinase, phosphorylated a tyrosine residue (Y105) on PKM2 itself. Further analysis revealed that this tyrosine residue was commonly phosphorylated in human cancers and that a mutant form of PKM2 lacking this tyrosine residue inhibited both “Warburg metabolism” and tumor growth. They thus propose that phosphorylation of PKM2 by oncogenic tyrosine kinases provides the very phosphotyrosine that binds to and inhibits PKM2 to induce the Warburg effect and promote tumor growth. The Warburg effect describes a pro-oncogenic metabolism switch such that cancer cells take up more glucose than normal tissue and favor incomplete oxidation of glucose even in the presence of oxygen. To better understand how tyrosine kinase signaling, which is commonly increased in tumors, regulates the Warburg effect, we performed phosphoproteomic studies. We found that oncogenic forms of fibroblast growth factor receptor type 1 inhibit the pyruvate kinase M2 (PKM2) isoform by direct phosphorylation of PKM2 tyrosine residue 105 (Y105). This inhibits the formation of active, tetrameric PKM2 by disrupting binding of the PKM2 cofactor fructose-1,6-bisphosphate. Furthermore, we found that phosphorylation of PKM2 Y105 is common in human cancers. The presence of a PKM2 mutant in which phenylalanine is substituted for Y105 (Y105F) in cancer cells leads to decreased cell proliferation under hypoxic conditions, increased oxidative phosphorylation with reduced lactate production, and reduced tumor growth in xenografts in nude mice. Our findings suggest that tyrosine phosphorylation regulates PKM2 to provide a metabolic advantage to tumor cells, thereby promoting tumor growth.

Tyrosine Phosphorylation of Mitochondrial Pyruvate Dehydrogenase Kinase 1 Is Important for Cancer Metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.

Fibroblast Growth Factor Receptor 3 Associates with and Tyrosine Phosphorylates p90 RSK2, Leading to RSK2 Activation That Mediates Hematopoietic Transformation

ABSTRACT Dysregulation of the receptor tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) plays a pathogenic role in a number of human hematopoietic malignancies and solid tumors. These include t(4;14) multiple myeloma associated with ectopic expression of FGFR3 and t(4;12)(p16;p13) acute myeloid leukemia associated with expression of a constitutively activated fusion tyrosine kinase, TEL-FGFR3. We recently reported that FGFR3 directly tyrosine phosphorylates RSK2 at Y529, which consequently regulates RSK2 activation. Here we identified Y707 as an additional tyrosine in RSK2 that is phosphorylated by FGFR3. Phosphorylation at Y707 contributes to RSK2 activation, through a putative disruption of the autoinhibitory αL-helix on the C terminus of RSK2, unlike Y529 phosphorylation, which facilitates ERK binding. Moreover, we found that FGFR3 interacts with RSK2 through residue W332 in the linker region of RSK2 and that this association is required for FGFR3-dependent phosphorylation of RSK2 at Y529 and Y707, as well as the subsequent RSK2 activation. Furthermore, in a murine bone marrow transplant assay, genetic deficiency in RSK2 resulted in a significantly delayed and attenuated myeloproliferative syndrome induced by TEL-FGFR3 as compared with wild-type cells, suggesting a critical role of RSK2 in FGFR3-induced hematopoietic transformation. Our current and previous findings represent a paradigm for tyrosine phosphorylation-dependent regulation of serine-threonine kinases.

Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells

An attractive target for therapeutic intervention is constitutively activated, mutant FLT3, which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing, and which is currently in late-stage clinical trials. However, the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel, structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here, we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487, which selectively targets mutant FLT3 protein kinase activity, is also shown to override PKC412 resistance in vitro, and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally, the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus, we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target, and which could potentially be used to override drug resistance in AML.

Target interaction profiling of midostaurin and its metabolites in neoplastic mast cells predicts distinct effects on activation and growth

Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multikinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC50 values. Midostaurin and CGP62221 also produced growth inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, which accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug competition experiments revealed that midostaurin interacts with KIT and several additional kinase targets. The key downstream regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE receptor downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation, which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs.

Cloning, expression and purification of a recombinant poly-histidine-linked HIV-1 protease

The gene coding for the HIV‐1 protease was cloned in an Escherichia coli expression vector adding three‐histidine codons to the amino and carboxy terminus of the protease sequence. Expression of the protease from this construct led to the accumulation of high amounts of insoluble histidine‐linked protease entrapped in inclusion bodies. The histidine‐linked protease could be efficiently released from purified inclusion bodies with 6 M guanidine hydrochloride and further purified by metal chelate affinity chromatography. The refolded protease cleaved synthetic peptide substrates and the viral polyprotein p55 with the same specificity as the wild type protease. It displays a specific activity of 4.4 μmol/min/mg.

Abstract 1257: Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Many tumor cells rely on aerobic glycolysis for their continued proliferation and survival, which is in part due to actively inhibited mitochondrial function. Myc and HIF-1 are believed to promote such inhibition by upregulating gene expression of pyruvate dehydrogenase kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial pyruvate dehydrogenase complex (PDC). However, how oncogenic signals activate PDHK1 to regulate cancer cell metabolism remains unclear. Here we report that oncogenic FGFR1 activates mitochondrial PDHK1 by tyrosine phosphorylation. FGFR1 directly phosphorylates PDHK1 at Y136, Y243 and Y244. Mutational, structural and biochemical studies revealed that phosphorylation at both Y243 and Y244, but not Y136 is required to promote ATP binding to PDHK1, which consequently facilitates PDHK1 binding to PDC scaffold to access substrate PDHA1. In contrast, Y136 phosphorylation may only function to enhance binding between PDHK1 and PDC. We also found that PDHK1 is commonly tyrosine phosphorylated by diverse oncogenic tyrosine kinases in different human cancers. Moreover, we generated cancer cells with stable knockdown of endogenous human PDHK1 and “rescue” expression of phosphorylation-deficient, catalytic hypomorph mouse PDHK1 mutants including Y134F and Y239/240F (mouse PDHK1 numberings correspond to human PDHK1 Y136F and Y243/244F, respectively). These “rescue” cancer cells demonstrated decreased cell proliferation under hypoxia, increased oxidative phosphorylation with decreased lactate production, and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation activates PDHK1 to inhibit mitochondrial function, providing a metabolic advantage for tumor growth. This represents a common, short-term molecular mechanism underlying the active inhibition of mitochondrial function in tumor cells, in addition to the chronic changes that are believed to be regulated by Myc and HIF-1. Moreover, inhibition of PDHK1 attenuates tumor growth, suggesting that PDHK1 may serve as a therapeutic target in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1257. doi:10.1158/1538-7445.AM2011-1257

Comparison of the Kinase Profile of Midostaurin (Rydapt) with That of Its Predominant Metabolites and the Potential Relevance of Some Newly Identified Targets to Leukemia Therapy

The multitargeted protein kinase inhibitor midostaurin is approved for the treatment of both newly diagnosed FLT3-mutated acute myeloid leukemia (AML) and KIT-driven advanced systemic mastocytosis. AML is a heterogeneous malignancy, and investigational drugs targeting FLT3 have shown disparate effects in patients with FLT3-mutated AML, probably as a result of their inhibiting different targets and pathways at the administered doses. However, the efficacy and side effects of drugs do not just reflect the biochemical and pharmacodynamic properties of the parent compound but are often comprised of complex cooperative effects between the properties of the parent and active metabolites. Following chronic dosing, two midostaurin metabolites attain steady-state plasma trough levels greater than that of the parent drug. In this study, we characterized these metabolites and determined their profiles as kinase inhibitors using radiometric transphosphorylation assays. Like midostaurin, the metabolites potently inhibit mutant forms of FLT3 and KIT and several additional kinases that either are directly involved in the deregulated signaling pathways or have been implicated as playing a role in AML via stromal support, such as IGF1R, LYN, PDPK1, RET, SYK, TRKA, and VEGFR2. Consequently, a complex interplay between the kinase activities of midostaurin and its metabolites is likely to contribute to the efficacy of midostaurin in AML and helps to engender the distinctive effects of the drug compared to those of other FLT3 inhibitors in this malignancy.

Abstract A140: Evaluation of prediction of in vivo activity from in vitro combinations: Examples using a MEK1/2 inhibitor combined with docetaxel in NSCLC models.

In an attempt to combat the resistance of tumors to chemotherapy, we have already described at this year9s AACR, the systematic evaluation of over 11,000 different compound combinations using the human cancer cell line encyclopedia. Correlations of synergy with genetic features were identified and some novel synergies discovered. Here, we describe the efficacy, tolerability and PK-PD of 23 different in vivo combinations selected from the screens to determine the predictability of in vitro screening, including two examples from the combination of a MEK1/2-inhibitor (MEK162) with the taxane, docetaxel. In vitro screens were conducted as previously described using 3-day viability assays, and inhibition of proliferation determined relative to untreated samples, and the degree of synergy scored using different types of analyses: Gaddum, Bliss, Loewe. Only those combinations showing synergy over a wide range of concentrations were chosen for in vivo study. For in vivo studies, cells were injected s.c. in the flank of athymic nude mice, and once tumors reached a mean size of at least 100 mm 3 were treated for 2-4 weeks with the appropriate dose and schedule of the compounds either as monotherapy, or in combination. Efficacy and tolerability were determined at the endpoint using the T/C TVol and T/C BW respectively to derive a combination-index as previously described by Clarke (1997), where a negative-value (-CCI) indicated synergy. In most cases, PK-PD was also measured in plasma and tumour either at steady-state and/or the endpoint to study the mechanism of the interaction and to check for drug-drug interactions and their eventual impact on PD and efficacy. Thus far, we have studied in vivo 13 different molecular targets across 6 different histotypes to give 23 different combinations. No antagonism was seen in vivo (+CCI), and 19/23 were deemed synergistic (CCI ≤-0.1), of which 8 showed regression which was not seen with the individual monotherapies. Of the 4 combinations showing no interaction, 2 were predicted by the in vitro score and the other 2 showed negative drug-drug interactions. There were no significant correlations between the CCI and the different types of in vitro score (p>0.35), but perhaps more importantly, cut-offs could be identified suggesting synergy could be predicted (p≤0.02) although not the extent of the interaction. Several novel combinations were identified for clinical investigation, including MEK162 combined with docetaxel in KRAS-mutant NSCLC, which in two different models in vivo had a CCI≤ -0.1, with PD-analyses showing that cytotoxic doses of the taxane activated the MAPK-pathway which was blocked by the combination. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A140. Citation Format: Paul Martin J. Mcsheehy, Alex Cao, Giordi Caponigro, David Duhl, Brant Firestone, Tom Gesner, Daniel Guthy, Jocelyn Holash, Fred King, Joseph Lehar, Christopher Leroy, Manway Liu, Lilli Petruzzelli, Dale Porter, Daniel Menezes, Anupama Reddy, Johannes Roesel, Christian Schnell, Timothy Smith, Mark Stump, Markus Wartmann, Marion Wiesmann. Evaluation of prediction of in vivo activity from in vitro combinations: Examples using a MEK1/2 inhibitor combined with docetaxel in NSCLC models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A140.

Abstract 2058: Systematic evaluation of drug combinations in vitro and in vivo.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC In recent years great advances have been made in developing targeted cancer therapeutics that produce dramatic responses in a subset of rationally selected patients. The initial breakthrough of the targeted design concept was established by the treatment of chronic myelogenous leukemia with Abl inhibitors and has been expanded to other cancer indications. As a consequence of this early success in CML, the identification and targeting of genetic lesions that confer cancer dependence has become an established strategy for drug discovery efforts. However, this approach has met with mixed degrees of success as confounding factors, such as tumor heterogeneity, have often resulted in partial responses and/or the emergence of resistance when targeted therapies were employed as single agents. To improve the therapeutic benefit in cancer, rationally-devised novel combinations of two or more agents are being explored clinically. To discover combinations that may be more effective therapies, an unbiased, systematic approach was used to identify drug combinations in vitro, using a panel of genetically diverse, and well characterized cell lines from the cancer cell line encyclopedia (CCLE: Barretina et al. Nature 2012). For three cancer indications, all pairwise combinations of 18 selected drugs (both novel inhibitors and standards of care) were tested as dose matrices in a proliferation assay. Synergistic interactions were scored using isobologram/Loewes excess inhibition and synergistic concentration ranges for each agent were identified. However, the clinical translation of positive combinations from in vitro matrix-based screens into clinically-relevant doses and schedules are challenging, due to host biology, tumor-stroma interactions, and the pharmacokinetic and pharmacodynamics of drug delivery. To explore this higher complexity, we evaluated the in vitro to in vivo translation of drug synergies in immune-compromised mouse tumor xenograft models. To recapitulate the pharmacological combination effects in vivo, mouse pharmacokinetic data and simulation was used to determine single agent doses that would result in the desired compound plasma concentration range and duration of action. Pharmakokinetics, pharmacodynamics, antitumor activity and tolerability of the combinations were then tested in tumor-bearing mice. Observed combination effects in vivo could in some cases be explained by either the expected biological pathway interactions or partially by physiological effects relating to drug-drug interactions. Citation Format: Marion Wiesmann, Mark Stump, Giordano Caponigro, David Duhl, Brant Firestone, Tom Gesner, Bjoern Gruenenfelder, Daniel Alexander Guthy, Jocelyn Holash, Fred King, Joseph Lehar, Christophe Leroy, Manway Liu, Lilli Petruzelli, Dale Porter, Paul McSheehy, Daniel Menezes, Anupama Reddy, Johannes Roesel, Christian Schnell, Timothy R. Smith, Markus Wartmann. Systematic evaluation of drug combinations in vitro and in vivo . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2058. doi:10.1158/1538-7445.AM2013-2058