Stefan Kaulfuss

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular d-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Mast cells in endometriosis: guilty or innocent bystanders?

Endometriosis (EMS) is a chronic, estrogen-dependent inflammatory disease characterized by growth of endometrial tissue outside the uterine cavity. Symptoms in EMS patients include severe pelvic pain, dysmenorrhea, dyspareunia and infertility. To date, medical therapies are mostly based on hormonal suppressive drugs that induce a hypoestrogenic state. Although being effective regarding the reduction of endometriotic tissue masses and pelvic pain, this treatment is accompanied by severe side effects. Since EMS is associated with chronic inflammation, novel therapeutic strategies also focus on immune modulating drugs. However, little is known about how and to what extent immune cell subsets contribute to the network of locally produced cytokines, chemokines and other mitogenic factors that modulate the growth of ectopic endometrial implants and the inflammation associated with them. Mast cells (MCs) are known to be key players of the immune system, especially during allergic reactions. However, in recent years MCs have been identified to exhibit a far broader range of functions and to be involved in host defense and wound healing responses. Here, recent reports that imply an involvement of MCs in EMS has been reviewed, while the value of novel mouse models for clarifying their contribution to the pathology of this condition has been discussed.

Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas.

Abstract 2645: BAY 1436032: A highly selective, potent and orally available inhibitor of mutant forms of IDH1

Isocitrate dehydrogenase 1 (IDH1) is a metabolic enzyme that is frequently mutated in certain cancers, with incidence rates ranging from 7-90% for glioma, chondrosarcoma, intrahepatic cholangiocarcinoma and AML. Wildtype IDH1 (wtIDH1) catalyzes the conversion of isocitrate to α-ketoglutarate (αKG), while tumor-associated mutant IDH1 (mIDH1) catalyzes a rogue reaction: the production of 2-hydroxyglutarate (2-HG) from αKG. 2-HG therefore represents an “oncometabolite” that is believed to play a role in cancer by interfering with αKG-dependent enzymes, which in turn causes hypermethylation of histones/DNA and a block of normal cellular differentiation. Mutant IDH1 is a “driver” oncogene and the inhibition of this altered enzyme will decrease the growth of mIDH1 dependent tumors. We report for the first time the preclinical profile and structure of BAY 1436032, a novel selective mIDH1 inhibitor. An optimization program based on a high throughput screening resulted in the identification of the clinical candidate BAY 1436032 for the treatment of mIDH1 dependent cancer. BAY 1436032 is a double-digit nanomolar and selective pan-inhibitor of the enzymatic activity of various IDH1-R132X mutants in vitro and displayed potent inhibition of 2-HG release (nanomolar range) in patient derived and engineered cell lines expressing different IDH1 mutants. In line with the proposed mode of action, a concentration-dependent lowering of 2-HG was observed in vitro accompanied by differentiation and maturation of mIDH1 tumor cells. Furthermore, BAY 1436032 showed a favourable selectivity profile against wtIDH1/2 and a large panel of off-targets in vitro. To the best of our knowledge we were able to show for the first time single agent in vivo efficacy in mIDH1 patient derived glioma and intrahepatic cholangiocarcinoma solid tumor models with this clinical candidate along with monitoring of intratumoral 2-HG levels as a predictive biomarker. The BBB penetration profile of BAY 1436032 is further supported by preclinical data on in vivo brain-plasma ratios. In conclusion, our data provide in vitro and in vivo proof of concept for BAY 1436032 as a potent and highly selective inhibitor of mutant forms of IDH1. The start of a Phase I study with BAY 1436032 is currently in preparation to determine the safety, tolerability, pharmacokinetics and preliminary anti-tumor and pharmacodynamic biomarker responses in patients with solid tumors. Citation Format: Olaf Panknin, Stefan Pusch, Lena Herbst, Stefan Kaulfuss, Katja Zimmermann, Hartmut Rehwinkel, Roland Neuhaus, Sven Ring, Michael Bruning, Claudia Stark, Katja Prelle, Martin Michels, Michael Jeffers, Holger Hess-Stumpp, Karl Ziegelbauer, Michael Brands, Alwin Kramer, Andreas von Deimling. BAY 1436032: A highly selective, potent and orally available inhibitor of mutant forms of IDH1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2645.

Abstract 1026: Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The receptor tyrosine kinase Tie2 is predominantly expressed in the endothelium but has also been identified on primitive hematopoietic stem cells, monocyte and macrophage subclasses, as well as on glioma or hematological tumor cells. Based on its expression in many patient-derived leukemic blasts inhibition of the Tie2 pathway may provide an attractive opportunity for therapeutic intervention in leukemias. In this study we report the pharmacological profile of a novel, highly potent and orally available Tie2 inhibitor (BAY-Tie2). The discovery and design process leading to BAY-Tie2 was performed with the goal of sparing other angiogenic RTKs, such as VEGFRs, FGFRs or PDGFRs. BAY-Tie2 is based on a novel imidazopyrazole core, combined with a SF5-substituted phenyl ring that fills the deep DFG-out pocket. BAY-Tie2 binds to Tie2 with a Kd value of 1.6 nM and is selective against VEGFR2 (Kd of 1600 nM), FGFR1 (<30% inhibition at 1 µM), FGFR2/3/4 (<10% inhibition at 1 µM) and PDGFRα/β (<30% inhibition at 100 nM). BAY-Tie2 potently inhibits Tie2 autophosphorylation in recombinant CHO-Tie2 and primary human umbilical vein endothelial cells (HUVEC) with IC50 values of 0.7 and 1.3 nM. Consistently, BAY-Tie2 was shown to inhibit Tie2 phosphorylation in vivo by analyzing angiopoietin-1 induced Tie2 phosphorylation status in extracts of murine lungs from BAY-Tie2-treated mice. In subcutaneous xenograft models of highly angiogenic tumors, BAY-Tie2 reduced tumor growth and showed evidence for potential combination benefit with anti-VEGF therapy. In order to explore the potential of a Tie2 inhibitor beyond affecting angiogenesis, we established disseminated leukemia models, using Tie2-expressing cell lines, such as the CML cell lines MEG-01 and EM-2. Both cell lines engrafted predominantly in bone marrow and spleen. Treatment started 3 days after i.v. cell implantation with either BAY-Tie2 or cytarabine and was well tolerated. Efficacy was monitored by a) inhibition of disease progression, b) weekly fluorescence-based in vivo imaging (IVI) using an Alexa750-labeled anti-human CD33 antibody, and c) q-RT-PCR specific for BCR-ABL and hCD45 in murine peripheral blood. BAY-Tie2 inhibited disease progression comparable to cytarabine. Tumor load measured by IVI was reduced in BAY-Tie2 treated groups by 45% in the MEG-01 and by 65% in the EM-2 model compared with the untreated control, very similar to the cytotoxic treatment with cytarabine. Quantitative RT-PCR on peripheral blood revealed that BAY-Tie2 and cytarabine delayed the appearance of circulating tumor cells in both CML models. These data demonstrate that BAY-Tie2 is an orally active Tie2 inhibitor that may have therapeutic benefit not only in angiogenic tumors but also in hematological, Tie2-expressing malignancies. Citation Format: Sylvia Gruenewald, Julia Schueler, Michael Haerter, Frank Suessmeier, Kerstin Klingner, Ulf Boemer, Stefan Kaulfuss, Alexander Walter, Mario Lobell, Ingo V. Hartung, Bernd Buchmann, Dieter Heldmann, Holger Hess-Stumpp, Karl Ziegelbauer. Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in 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 1026. doi:10.1158/1538-7445.AM2014-1026

Abstract 1049: Pharmacological characterization of a novel potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with robust in vivo efficacy and increased therapeutic index with niacin supplementation

Nicotinamide adenine dinucleotide (NAD+) is an important metabolite and cofactor for a number of cellular processes including genomic stability and epigenetic regulation. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate limiting enzyme in NAD+ salvaging from nicotinamide. Many tumor cells have an increased need for NAD+ and are therefore highly sensitive to NAMPT inhibition. We have developed a selective and potent small molecule inhibitor, Cmpd1, which inhibits the NAMPT enzyme with sub-nanomolar potency. In cells, Cmpd1 treatment leads to an almost complete reduction of both NAD- and ATP-levels, followed by the induction of cell death. In order to identify sensitive cancer subtypes and associated biomarkers, we analyzed cell sensitivity to NAMPT inhibition in a panel of 240 cell lines. Small cell lung cancer and hematological malignancies were particularly sensitive to NAMPT inhibition. Overall, expression levels of NAMPT and NAD+ consuming enzymes correlated well with sensitivity to NAMPT inhibition. In order to investigate the effect of niacin on the antiproliferative effect of NAMPT inhibition across the cell panel, we further analyzed the cell sensitivity in the presence of niacin. Cells with a functional Preiss-Handler pathway can generate NAD+ from niacin, independent of NAMPT. We found that the antiproliferative effect of Cmpd1 was neutralized by niacin in 60% of the analyzed cells. The mRNA levels of nicotinate phosphoribosyltransferase 1 (NAPRT1), a central enzyme of the Preiss-Haendler pathway, predicted well the niacin rescue status of the cells. In vivo, niacin supplementation led to an at least 10-fold increase in the maximum tolerated dose of Cmpd1 in mice. Antitumor efficacy of Cmpd1 was abolished by niacin supplementation in xenografts derived from NAPRT1 expressing cells, but not in NAPRT1-deficient models. These data demonstrate that the therapeutic index of NAMPT inhibitors may be increased in NAPRT1-deficient tumors by niacin supplementation. Citation Format: Maria Quanz, Claudia Merz, Andreas Bernthaler, Stefan Kaulfuss, Anja Richter, Marcus Bauser, Andrea Haegebarth. Pharmacological characterization of a novel potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with robust in vivo efficacy and increased therapeutic index with niacin supplementation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1049.