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The lactate transporter SLC16A1/monocarboxylate transporter 1 (MCT1) plays a central role in tumor cell energy homeostasis. In a cell-based screen, we identified a novel class of MCT1 inhibitors, including BAY-8002, which potently suppress bidirectional lactate transport. We investigated the antiproliferative activity of BAY-8002 in a panel of 246 cancer cell lines and show that hematopoietic tumor cells, in particular diffuse large B-cell lymphoma cell lines, and subsets of solid tumor models are particularly sensitive to MCT1 inhibition. Associated markers of sensitivity were, among others, lack of MCT4 expression, low pleckstrin homology like domain family A member 2, and high pellino E3 ubiquitin protein ligase 1 expression. The antitumor effect of MCT1 inhibition was less pronounced on tumor xenografts, with tumor stasis being the maximal response. BAY-8002 significantly increased intratumor lactate levels and transiently modulated pyruvate levels. In order to address potential acquired resistance mechanisms to MCT1 inhibition, we generated MCT1 inhibitor–resistant cell lines and show that resistance can occur by upregulation of MCT4 even in the presence of sufficient oxygen, as well as by shifting energy generation toward oxidative phosphorylation. These findings provide insight into novel aspects of tumor response to MCT1 modulation and offer further rationale for patient selection in the clinical development of MCT1 inhibitors. Mol Cancer Ther; 17(11); 2285–96. ©2018 AACR.

Supplementary Methods: Synthesis and characterization of BAY-8002; Tritium labeling of AZD3965 and BAY-8002; MCT1 and MCT4 14C-lactate uptake measurements in the X. laevis oocytes expression System

Supplemental Table S1: Selectivity profile of BAY-8002. Supplemental Table S2: Summary table of functional and proliferation IC50 data for BAY-8002 and AZD3965. Supplemental Table S3: Responses to BAY-8002 and AZD3965 of cell lines with acquired resistance to MCT1 inhibition.

Supplemental Figure S1: A, SLC16A1 (MCT1), SLC16A7 (MCT2), SLC16A8 (MCT3) and SLC16A3 (MCT4) mRNA expression levels and Effects of BAY-8002 on lactate Export and ECAR. Supplemental Figure S2: Binding affinity of the two MCT1 inhibitors BAY-8002 and AZD3965. Supplemental Figure S3: supplemental cell panel analysis. Supplemental Figure S4: subset analysis of MCT4 low expressing cells from the cell panel Analysis. Supplemental Figure S5: Intratumor 2/3-PG and 6PG levels after treatment with AZD3965

Supplementary Data from Small-Molecule Drugs Mimicking DNA Damage: A New Strategy for Sensitizing Tumors to Radiotherapy

The lactate transporter SLC16A1/monocarboxylate transporter 1 (MCT1) plays a central role in tumor cell energy homeostasis. In a cell-based screen, we identified a novel class of MCT1 inhibitors, including BAY-8002, which potently suppress bidirectional lactate transport. We investigated the antiproliferative activity of BAY-8002 in a panel of 246 cancer cell lines and show that hematopoietic tumor cells, in particular diffuse large B-cell lymphoma cell lines, and subsets of solid tumor models are particularly sensitive to MCT1 inhibition. Associated markers of sensitivity were, among others, lack of MCT4 expression, low pleckstrin homology like domain family A member 2, and high pellino E3 ubiquitin protein ligase 1 expression. The antitumor effect of MCT1 inhibition was less pronounced on tumor xenografts, with tumor stasis being the maximal response. BAY-8002 significantly increased intratumor lactate levels and transiently modulated pyruvate levels. In order to address potential acquired resistance mechanisms to MCT1 inhibition, we generated MCT1 inhibitor–resistant cell lines and show that resistance can occur by upregulation of MCT4 even in the presence of sufficient oxygen, as well as by shifting energy generation toward oxidative phosphorylation. These findings provide insight into novel aspects of tumor response to MCT1 modulation and offer further rationale for patient selection in the clinical development of MCT1 inhibitors. Mol Cancer Ther; 17(11); 2285–96. ©2018 AACR.

Despite the recent advances in the treatment of ovarian cancer, it remains an area of high unmet medical need. Epithelial ovarian cancer is associated with high levels of mesothelin expression, and therefore, mesothelin is an attractive candidate target for the treatment of this disease. Herein, we investigated the antitumor efficacy of the mesothelin-targeting antibody-drug conjugate (ADC) anetumab ravtansine as a novel treatment option for ovarian cancer in monotherapy and in combination with the antitumor agents pegylated liposomal doxorubicin (PLD), carboplatin, copanlisib and bevacizumab. Anetumab ravtansine showed potent antitumor activity as a monotherapy in ovarian cancer models with high mesothelin expression. No activity was seen in mesothelin-negative models. The combination of anetumab ravtansine with PLD showed additive anti-proliferative activity in vitro, which translated into improved therapeutic in vivo efficacy in ovarian cancer cell line- and patient-derived xenograft (PDX) models compared to either agents as a monotherapy. The combination of anetumab ravtansine with the PI3Kα/δ inhibitor copanlisib was additive in the OVCAR-3 and OVCAR-8 cell lines in vitro, showing increased apoptosis in response to the combination treatment. In vivo, the combination of anetumab ravtansine with copanlisib resulted in more potent antitumor activity than either of the treatments alone. Likewise, the combination of anetumab ravtansine with carboplatin or bevacizumab showed improved in vivo efficacy in the ST081 and OVCAR-3 models, respectively. All combinations were well-tolerated. Taken together, these data support the development of anetumab ravtansine for ovarian cancer treatment and highlight its suitability for combination therapy with PLD, carboplatin, copanlisib, or bevacizumab.

MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival.

Nicotinamide phosphoribosyl-transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway, generating nicotinamide adenine dinucleotide (NAD) from nicotinamide (NAM). Inhibition of intracellular NAMPT activity represents a differentiated mode-of-action for tumor-targeting antibody-drug conjugates (ADCs) as it is not dependent on cell proliferation. Thus, NAMPT inhibitor-based ADCs have the potential to target both proliferating and resting tumor cells. We therefore developed a novel structural class of NAMPT inhibitors (NAMPTi) as a potent ADC payload class and characterized NAMPTi and NAMPTi-ADCs in vitro and in vivo in preclinical tumor models. We profiled the small molecule NAMPTi BAY-346 in comparison to the kinesin spindle protein inhibitor (KSPi) BAY-331 on a panel of 350 cancer cell lines from various tumor indications showing a differential sensitivity profile of BAY-346 vs BAY-331, with cell lines characterized by low NAMPT mRNA levels as being very sensitive to BAY-346. NAMPTi BAY-346, but not KSPi BAY-331, reduced the viability of quiescent HaCat cells as well as of serum starved non-proliferating NCI-N87 cells. In vitro treatment with NAMPTi BAY-346, which bears a pyridine warhead that can be phosphoribosylated by NAMPT, resulted in IC50 values in the nanomolar to subnanomolar range (2.8 nM to 0.01 nM) in cell lines derived from solid and hematologic tumor indications (e.g., THP-1, MV-4-11, U-251, NCI-H292, MDA-MB-453, LoVo, KPL4, HT1197 and BxPC3). BAY-346 was 100-fold more potent than the small molecule NAMPTi BAY-248, which cannot be phosphoribosylated. A BAY-346 derived NAMPTi was conjugated as a payload to a series of antibodies targeting different tumor-associated antigens: C4.4a (LYPD3), HER2, B7H3 (CD276), and TWEAKR (Fn14/ TNFRSF10A). The resulting NAMPTi-ADCs were tested in proliferation and cellular mechanistic in vitro assays. NAMPTi-ADCs depleted NAD+ in tumor cells and showed potent growth inhibitory activity with IC50 values in the subnanomolar- to nanomolar range in a target-dependent manner. A C4.4a-NAMPTi-ADC and a HER2-NAMPTi-ADC were tested In the C4.4a- and HER2-expressing MDA-MB-453 cell line derived subcutaneous breast cancer model xenografted on NOD/SCID mice. Both NAMPTi-ADCs showed highly potent anti-tumor Efficacy: The C4.4a-NAMPTi-ADC induced complete responses (in 3 of 8 mice) and stable diseases (in 5 of 8 mice) and the HER2-NAMPTi-ADC achieved complete tumor regression in all treated animals. In addition, in the THP-1 acute myeloid leukemia (AML) subcutaneous in vivo model, a B7H3-NAMPTi-ADC induced complete tumor responses in 7 of 8 treated animals. Taken together, we identified a new series of NAMPT inhibitors as a novel class of ADC payloads exhibiting strong in vivo efficacy in various preclinical xenograft models. Citation Format: Anette Sommer, Stefanie Hammer, Sandra Berndt, Antje M. Wengner, Niels Boehnke, Markus Berger, Nils Griebenow, Andreas Steffen, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Anja Giese, Maria Quanz, Zhengzheng Bao, Xiuli Wu, Hilmar Weinmann, Lars Linden, Bertolt Kreft, Dominik Mumberg. Anti-tumor activity of a novel structural class of NAMPT inhibitor-based ADCs in models of hematologic and solid tumor indications [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1807.

Melanomas are highly radioresistant tumors, mainly due to efficient DNA double-strand break (DSB) repair. Dbait (which stands for DNA strand break bait) molecules mimic DSBs and trap DNA repair proteins, thereby inhibiting repair of DNA damage induced by radiation therapy (RT). First, the cytotoxic efficacy of Dbait in combination with RT was evaluated in vitro in SK28 and 501mel human melanoma cell lines. Though the extent of RT-induced damage was not increased by Dbait, it persisted for longer revealing a repair defect. Dbait enhanced RT efficacy independently of RT doses. We further assayed the capacity of DT01 (clinical form of Dbait) to enhance efficacy of "palliative" RT (10 × 3 Gy) or "radical" RT (20 × 3 Gy), in an SK28 xenografted model. Inhibition of repair of RT-induced DSB by DT01 was revealed by the significant increase of micronuclei in tumors treated with combined treatment. Mice treated with DT01 and RT combination had significantly better tumor growth control and longer survival compared to RT alone with the "palliative" protocol [tumor growth delay (TGD) by 5.7-fold; median survival: 119 vs 67days] or the "radical" protocol (TGD by 3.2-fold; median survival: 221 vs 109days). Only animals that received the combined treatment showed complete responses. No additional toxicity was observed in any DT01-treated groups. This preclinical study provides encouraging results for a combination of a new DNA repair inhibitor, DT01, with RT, in the absence of toxicity. A first-in-human phase I study is currently under way in the palliative management of melanoma in-transit metastases (DRIIM trial).

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.

Ovarian cancer remains an area of high unmet medical need, with 239,000 patients newly diagnosed per year. Here we describe the mesothelin-targeting antibody drug-conjugate anetumab ravtansine as a novel treatment option for ovarian cancer. Internalization of anetumab ravtansine and co-localization with lysosomal markers in ovarian cancer cells was accompanied by rapid resynthesis of mesothelin, which may allow consecutive anetumab ravtansine treatment cycles. The strong antitumor activity of anetumab ravtansine was preserved during repeated treatment cycles in the OVCAR-3 ovarian cancer in vivo model. Mechanistically, treatment with anetumab ravtansine caused mitotic arrest characterized by increased pHH3 signal and monopolar spindle structures. Interestingly, anetumab ravtansine monotherapy also induced DNA damage as indicated by focal γH2AX signals. Ultimately, the cells engaged in apoptotic cell death. Strong in vitro monotherapy activity was demonstrated in a set of 11 ovarian cancer cell lines with IC50s between 3 and 90nM. The in vivo efficacy of anetumab ravtansine dosed at 2.5 mg/kg Q3Dx3 i.v. anetumab ravtansine was evaluated in 9 human ovarian cancer models with varying mesothelin expression levels. Strong in vivo efficacy at this moderate dose with T/C Citation Format: Christoph A. Schatz, Maria Quanz, Urs B. Hagemann, Sabine Zitzmann-Kolbe, Beatrix Stelte-Ludwig, Sven Golfier, Charlotte Kopitz, Cem Elbi, Karl Ziegelbauer, Dominik Mumberg. Anetumab ravtansine has single-agent activity in mesothelin-expressing human ovarian cancer models and potentiates the activity of chemotherapeutics and targeted agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1780.

Purpose: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage. Experimental Design: A novel strategy for inhibiting DNA repair was tested. We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait) and act by disorganizing damage signaling and DNA repair. We analyzed the effects of Dbait in cultured cells and on xenografted tumors growth and performed preliminary studies of their mechanism(s) of action. Results: The selected Dbait molecules activate H2AX phosphorylation in cell culture and in xenografted tumors. In vitro, this activation correlates with the reduction of Nijmegen breakage syndrome 1 and p53-binding protein 1 repair foci formation after irradiation. Cells are sensitized to irradiation and do not efficiently repair DNA damage. In vivo, Dbait induces regression of radioresistant head and neck squamous cell carcinoma (Hep2) and melanoma (SK28 and LU1205) tumors. The combination of Dbait32Hc treatment and fractionated radiotherapy significantly enhanced the therapeutic effect. Tumor growth control by Dbait molecules depended directly on the dose and was observed with various irradiation protocols. The induction of H2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of “false” DNA damage signaling and repair inhibition. Conclusions: These data validate the concept of introducing small DNA molecules, which mimic DNA damage, to trigger “false” signaling of DNA damage and impair DNA repair of damaged chromosomes. This new strategy could provide a new method for enhancing radiotherapy efficiency in radioresistant tumors.

Malignant transformation is accompanied by increased reactive oxygen species (ROS) known to promote carcinogenesis and damage free nucleotides and DNA. During replication, damaged nucleotides are incorporated into DNA resulting in DNA breaks and mutations, which can ultimately lead to cell death. Cancer cells may evade this process via overexpression of MTH1 (also known as NUDT1), a member of nudix phosphohydrolase protein family, which converts the oxidized nucleotides 8-oxo-dGTP and 2-OH-dATP into the corresponding monophosphates thus preventing their incorporation into DNA and avoiding cell death. Initial RNAi-mediated knockdown of MTH1 and tool compounds (TH588, (S)-crizotinib) inhibiting MTH1 supported this model. As MTH1 is not essential for non-transformed cell survival, MTH1 was hypothesized to be a non-oncogenic cancer addiction and a potential broad-spectrum cancer target. Attractive target rationale combined with previous success in identifying potent and cellularly active MTH1 inhibitors prompted us to develop new cancer therapeutics inhibiting MTH1. By using fragment-based screening and structure-based drug design, a series of 4-amino-2-carboxamide-7-azaindoles was identified. We developed biochemically potent and selective MTH1 inhibitors with good cell permeability and metabolic stability. These MTH1 inhibitors demonstrated target engagement in cellular thermal shift assay (CETSA), and a strong positive correlation between cellular and biochemical potency was observed. One promising MTH1 inhibitor from this structural class was BAY-707. Unexpectedly however, these properties did not translate into accumulation of oxidized nucleotides within DNA and consequent induction of γH2AX and DNA damage response. Moreover, while tool compounds (TH588, (S)-crizotinib) were confirmed to be biochemically potent MTH1 inhibitors which stunted the proliferation of a range of cancer cell lines, our more potent and cellularly active MTH1 inhibitors, including BAY-707, demonstrated no significant effect on cancer cell survival. Furthermore, we were unable to demonstrate in vivo efficacy using xenograft models of human cancers or syngeneic mouse tumor models. Finally, our in vitro and in vivo combination studies with pro-oxidants, standard-of-care drugs or radiation also failed to result in significant additive or synergistic growth inhibitory effects on cancer cells. Thus, our findings support the recently published observations made with other potent and selective MTH1 chemical probes (AZ compound 15, IACS-4759, NPD7155) and CRISPR/Cas9-mediated MTH1 knockout. Based on these observations and our additional target validation experiments, we concluded that MTH1 is not essential for cancer cell survival or for the sanitization of damaged nucleotides within cells and thus not a viable target for development of novel anticancer agents. Citation Format: Manuel Ellermann, Anja Giese, Ashley Eheim, Stefanie Bunse, Roland Neuhaus, Jörg Weiske, Maria Quanz, Andrea Glasauer, Fredrik Rahm, Jenny Viklund, Martin Andersson, Tobias Ginman, Rickard Forsblom, Johan Lindström, Lionel Trésaugues, Matyas Gorjanacz. Novel class of potent and selective inhibitors efface MTH1 as broad-spectrum cancer target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5226. doi:10.1158/1538-7445.AM2017-5226

We discovered that exogenous double-stranded DNA molecules are actively transported through the cytoplasm of eukaryotic cells. This transport is related to the activity of molecular motors belonging to the kinesin-14 family: KIFC1 and NCD.This result was achieved through in cellula single molecule experiments, which allowed us observing the motion of the naked DNA molecules in HeLa cells, in real time. These experiments were complemented by mass spectroscopy measurements to isolate the motor(s) responsible for this active transport. Eventually, the NCD driven transport of dsDNA molecules was reproduced in an in-vitro minimal system.

International audience; During the past years, exogenous DNA molecules have been used in gene and molecular therapy. At present, it is not known how these DNA molecules reach the cell nucleus. We used an in cell single-molecule approach to observe the motion of exogenous short DNA molecules in the cytoplasm of eukaryotic cells. Our observations suggest an active transport of the DNA along the cytoskeleton filaments. We used an in vitro motility assay, in which the motion of single-DNA molecules along cytoskeleton filaments in cell extracts is monitored; we demonstrate that microtubule-associated motors are involved in this transport. Precipitation of DNA-bound proteins and mass spectrometry analyses reveal the preferential binding of the kinesin KIFC1 on DNA. Cell extract depletion of kinesin KIFC1 significantly decreases DNA motion, confirming the active implication of this molecular motor in the intracellular DNA transport.

DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr-7) residue of heat shock protein 90α (Hsp90α) was phosphorylated immediately after DNA damage. The phosphorylated Hsp90α then accumulated at sites of DNA double strand breaks and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90α was dependent on phosphatidylinositol 3-kinase-like kinases, including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DNA double strand breaks by nonhomologous end-joining and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90α. In vivo, reverse phase protein array data for tumors revealed that basal levels of Thr-7-phosphorylated Hsp90α were correlated with phosphorylated histone H2AX levels. The Thr-7 phosphorylation of the ubiquitously produced and secreted Hsp90α may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone.

Cells respond to DNA damage by activating an intricate signaling network leading to DNA repair, cell cycle arrest or apoptosis. In recent years, progress has been made in the discovery and characterization of a number of DNA repair pathways, and it has become apparent that the inhibition of specific components of these pathways could offer new targets for combating the resistance of tumors to chemotherapy or radiotherapy. A thorough understanding of the various DNA repair pathways and their regulation is therefore essential. The DNA damage response (DDR) is of great importance in determining cell fate decisions. It includes many signal amplification steps and several steps that are partly redundant due to the ability of different kinases to phosphorylate the same target. Furthermore, the timing and origin of the damage play an important role in determining the DNA repair pathway activated. All this makes it difficult to study the role of one particular protein in DNA damage signaling. In addition, the available tools for activating DNA repair pathways are mostly agents that systematically produce more than one type of DNA damage. Even if the damage caused is initially of one predominant type (as for topoisomerase inhibitors, alkylators or the I-SceI endonuclease system), the damage may rapidly be transformed by normal cellular processes, such as DNA replication, or specific nuclease activities. Studies of the DDR become evenmore complicated if the agent used to create DNA lesions also damages other cellular components, as is the case for ionizing radiation (IR), alkylators and hydrogen peroxide. Furthermore, the damage is transient, as DNA damage signaling is rapid and lesions are quickly repaired. The signal induced by the damage therefore disappears rapidly, soon after the induction of damage. In some cells, the DNA may not be successfully repaired, leading to apoptosis or senescence. These aspects make it difficult to study the signaling network induced by a given type of damage. In this chapter, we will provide an overview of the response of the cell to DNA damage and possible ways of inducing a DDR in cells without actually damaging chromatin. We will focus on stabilized short interfering DNAmolecules (siDNA), which mimic different types of damage and induce a pure damage-specific response. SiDNA and Other Tools for the Indirect Induction of DNA Damage Responses

Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an “all-or-none” pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a “false” DNA damage signaling that disorganizes the DNA repair system.

Malignant cells are known for their accelerated metabolism, high energy requirements, and increased glucose uptake. They are characterized by high rates of glycolysis and metabolize glucose into lactate even under aerobic conditions, a hallmark of cancer known as the Warburg effect. Transport of glucose across the plasma membrane is the first and rate-limiting step for glucose metabolism and is mediated among others by facilitative glucose transporter (GLUT) proteins. Among the 14 GLUT proteins, GLUT1 and to a lesser degree GLUT3 are up regulated in many tumors. GLUT1 is also a prime target for the transcription factor hypoxia-inducible factor (HIF)-1α. Close relationships between GLUT1 expression, tumor development and progression, as well as poor overall survival have been described for several tumor entities. We have developed a GLUT1-selective, potent small molecule inhibitor, BAY-GLUT1, which inhibits both glucose uptake and ATP generation in GLUT1-expressing DLD-1 tumor cells in a glucose competitive way with IC50s of 4 and 3 nM, respectively. Selectivity was tested in either GLUT1-deficient DLD-1 cells or GLUT2/4-overexpressing recombinant CHO cells. In this study we report on the metabolic reaction of two adenocarcinoma cell lines, HeLa-MaTu (cervix) and DLD-1 (colon), to BAY-GLUT1 treatment. The two cell lines were treated with 30 nM BAY-GLUT1 for 6 and 24 hours under normoxic conditions and investigated by Metanomics Health's untargeted, broad metabolite profiling platform (MxP® Broad Profiling) to identify metabolic mechanisms explaining the differential susceptibility of cancer cells towards BAY-GLUT1. Hereby, two types of mass spectrometry (gas chromatography-mass spectrometry and liquid chromatography-MS/MS) analysis were applied to evaluate levels of 215 intracellular metabolites covering major metabolic pathways. Both inhibitor-treated cell lines showed dramatic decreases of glucose-6-phosphate and other glycolysis-related metabolites indicating efficient reduction in glycolytic activity. Subsequently, levels of several TCA cycle intermediates were also reduced. Our metabolic findings might suggest that DLD-1 preferentially uses glutamate and its precursor metabolites proline and 5-oxoproline to replenish the TCA cycle at the level of alpha-ketoglutarate. Interestingly, due to a gain-of-function mutation of isocitrate dehydrogenase, DLD-1 cells are able to reduce alpha-ketoglutarate into 2-hydroxyglutaric acid which might play an important role in controlling stability of transcription factor HIF-1α and in inducing metabolic reprogramming. Understanding the metabolic pathway changes upon GLUT inhibition could help to identify predictive conditions and/or biomarkers for treatment response. Citation Format: Sylvia Gruenewald, Ulrike Rennefahrt, Sandra G. Maldonado, Alexander Walter, Heike Petrul, Mélanie Héroult, Iring Heisler, Maria Quanz, Patrick Steigemann, Bernd Buchmann, Andrea Haegebarth. Metabolic responses in cancer cells with differential susceptibility to GLUT1 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1164. doi:10.1158/1538-7445.AM2015-1164

Malignant cells are known for their accelerated metabolism, high energy requirements, and increased glucose uptake. Transport of glucose across the plasma membrane is the first and rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose uptake in malignant cells has been associated with upregulated expression of glucose transporters, mainly overexpression of GLUT1 and/or GLUT3. There is limited knowledge about how selective (e.g. GLUT1) versus broad (multi-) GLUT inhibition affects glucose homeostasis in tumor bearing mice. Using potent small molecule inhibitors, we compared [14C]-2-Deoxy-D-Glucose (2-DG) distribution after selective GLUT1 versus GLUT1, 3 and 4 [multi-GLUT] inhibition and versus control in human NSCLC NCI-H460 tumor bearing mice. A single dose of a GLUT1 selective and a multi-GLUT inhibitor were administered to NCI-H460 tumor bearing NMRI nu/nu mice. At the respective Cmax concentrations, a bolus of 2-DG was rapidly injected intra-peritoneally and the distribution of metabolically stable 2-DG was obtained using whole-body autoradiography after 15 min and 120 min. With the multi-GLUT inhibitor only a very short inhibition of 2-DG uptake was observed in the NCI-H460 tumors while a long-lasting inhibition was detected in heart, brain and brown fat tissue. In contrast, a long-lasting inhibition of 2-DG uptake was observed in NCI-H460 tumors for the selective GLUT1 inhibitor. 2-DG concentrations were reduced in the brain following administration of the selective GLUT1 inhibitor at 15 min and returned to normal levels at 120 min while the tumor 2-DG concentration stayed low. The 2-DG findings go in parallel with the histopathological findings present in the brain and heart after treatment with the multi-GLUT inhibitor. However, similar histopathological findings have not been observed in the brain and heart after treatment with the selective GLUT-1 inhibitor. Therefore, selective GLUT1 inhibition is associated with a sustained low 2-DG concentration in the NCI-H460 tumors while only minor changes in glucose homeostasis were observed in other organ systems. Citation Format: Melanie Heroult, Wolfram Steinke, Anna-Lena Frisk, Sandra Borkowski, Kirstin Meyer, Heike Petrul, Iring Heisler, Maria Quanz, Roland Neuhaus, Bernd Buchmann, Thomas Mueller, Marcus Bauser, Andrea Haegebarth, Michael Brands, Karl Ziegelbauer. Effects of selective and broad glucose transporter (GLUT) inhibition on glucose distribution in tumor bearing mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1442. doi:10.1158/1538-7445.AM2014-1442

The DNA-dependent protein kinase (DNA-PK) may function as a key signaling kinase in various cellular pathways other than DNA repair. Using a two-dimensional gel electrophoresis approach and stable DNA double-strand break-mimicking molecules (Dbait32Hc) to activate DNA-PK in the nucleus and cytoplasm, we identified 26 proteins that were highly phosphorylated following DNA-PK activation. Most of these proteins are involved in protein stability and degradation, cell signaling and the cytoskeleton. We investigated the relationship between DNA-PK and the cytoskeleton and found that the intermediate filament (IF) vimentin was a target of DNA-PK in vitro and in cells. Vimentin was phosphorylated at Ser459, by DNA-PK, in cells transfected with Dbait32Hc. We produced specific antibodies and showed that Ser459-P-vimentin was mostly located at cell protrusions. In migratory cells, the vimentin phosphorylation induced by Dbait32Hc was associated with a lower cellular adhesion and migration capacity. Thus, this approach led to the identification of downstream cytoplasmic targets of DNA-PK and revealed a connection between DNA damage signaling and the cytoskeleton.

Tumor cells often display increased genomic instability and many anticancer treatments use genotoxic agents targeting DNA. Therefore DNA damage response biomarkers, such as the phosphorylated form of the histone H2AX (?-H2AX), are useful for both tumor characterization and the evaluation of chemotherapy efficacy. However, a major drawback for their use in a clinical setting is their limited accessibility. The molecular chaperone Heat shock protein 90a (Hsp90a), a target of new therapeutic cancer strategies, is often elevated in cancer and has been detected in the serum of patients with various cancer types. Hsp90a represents thus an accessible biomarker for some cancer evaluation. We present here evidences that a phosphorylated form of Hsp90a is a potential surrogate biomarker to follow response to DNA damage induced by treatments in tumours. We recently found that DNA-PK phosphorylates Hsp90a in response to ionizing radiation (P-Thr7-Hsp90a) [1]. Basal levels of P-Thr7-Hsp90a correlated with ?-H2AX levels in a variety of human xenografted tumors in mice. We have previously shown that the treatment with short double-stranded DNA molecules (named Dbait) impairs the repair of irradiation-induced DNA damage in tumors [2,3]. By mimicking DNA double-strand breaks (DSBs), Dbait molecules activate the DNA-dependent protein kinase (DNA-PK), a central DSB signaling enzyme. Dbait treatment induces both nuclear and cytoplasmic phosphorylation of Hsp90a. Moreover, we were able to detect the Thr7 phosphorylated form in the cell culture media of Hsp90a-secreting tumor cells. The P-Thr7-Hsp90a biomarker may therefore represent a surrogate biomarker of genomic instability and DNA damage, allowing both the stratification of cancer patients and monitoring of genotoxic drug responses. 1. Quanz et al (2012) J Biol Chem287, 8803-8815. 2. Quanz et al (2009) Clin Cancer Res15, 1308-1316. 3. Quanz et al (2009) PLoS One4, e6298. Source of funding: Institut Curie, CNRS, INSERM, Agence National de le Recherche: Grant ANR-08-Biot-009-02.