Molecular basis of GDF15 induction and suppression by drugs in cardiomyocytes and cancer cells toward precision medicine.

GDF15 has recently emerged as a key driver of the development of various disease conditions including cancer cachexia. Not only the tumor itself but also adverse effects of chemotherapy have been reported to contribute to increased GDF15. Although regulation of GDF15 transcription by BET domain has recently been reported, the molecular mechanisms of GDF15 gene regulation by drugs are still unknown, leaving uncertainty about the safe and effective therapeutic strategies targeting GDF15. We screened various cardiotoxic drugs and BET inhibitors for their effects on GDF15 regulation in human cardiomyocytes and cancer cell lines and analyzed in-house and public gene signature databases. We found that DNA damaging drugs induce GDF15 in cardiomyocytes more strongly than drugs with other modes of action. In cancer cells, GDF15 induction varied depending on drug- and cell type-specific gene signatures including mutations in PI3KCA, TP53, BRAF and MUC16. GDF15 suppression by BET inhibition is particularly effective in cancer cells with low activity of the PI3K/Akt axis and high extracellular concentrations of pantothenate. Our findings provide insights that the risk for GDF15 overexpression and concomitant cachexia can be reduced by a personalized selection of anticancer drugs and patients for precision medicine.

Therapeutic impact of BET inhibitor BI 894999 treatment: backtranslation from the clinic.

BACKGROUND: BET inhibitors have been tested in several clinical trials where, despite encouraging preclinical results, substantial clinical benefit in monotherapy remains limited. This work illustrates the translational challenges and reports new data around the novel BET inhibitor, BI 894999. At clinically achievable concentrations, mechanistic studies were carried out to study pathway modulation and rational drug combinations. METHODS: BRD-NUT fusions are oncogenic drivers in NUT carcinoma (NC). The effects of BI 894999 on proliferation, chromatin binding and pathway modulation were studied in NC in vitro. These studies were complemented by efficacy studies either as a single agent or in combination with the clinical p300/CBP inhibitor CCS1477. RESULTS: Based on the modelling of preclinical and clinical data, we proposed and implemented a new clinical scheduling regimen. This led to plasma levels sufficient to fully dislodge BRD-NUT from chromatin and to sustained and pronounced pharmacodynamic (PD) modulation of HEXIM1 and HIST2H2BF. Platelet counts in patient blood samples were improved compared to previous schedules. Rational combination studies of BI 894999 performed at clinically meaningful concentrations led to tumour regressions in all NC xenograft models tested. CONCLUSIONS: BI 894999 holds significant potential as a combination drug and CCS1477 p300/CBP inhibitor is a promising partner for future clinical trials.

Report of the First International Symposium on NUT Carcinoma.

NUT carcinoma is a rare, aggressive cancer defined by rearrangements of the NUTM1 gene. No routinely effective treatments of NUT carcinoma exist, despite harboring a targetable oncoprotein, most commonly BRD4-NUT. The vast majority of cases are fatal. Poor awareness of the disease is a major obstacle to progress in the treatment of NUT carcinoma. While the incidence likely exceeds that of Ewing sarcoma, and BRD4-NUT heralded the bromodomain and extra-terminal domain (BET) inhibitor class of selective epigenetic modulators, NUT carcinoma is incorrectly perceived as "impossibly rare," and therefore receives comparatively little private or governmental funding or prioritization by pharma. To raise awareness, propagate scientific knowledge, and initiate a consensus on standard and targeted treatment of NUT carcinoma, we held the First International Symposium on NUT Carcinoma on March 3, 2021. This virtual event had more than eighty attendees from the Americas, Europe, Asia, and Australia. Patients with NUT carcinoma and family members were represented and shared perspectives. Broadly, the four areas discussed by experts in the field included (1) the biology of NUT carcinoma; (2) standard approaches to the treatment of NUT carcinoma; (3) results of clinical trials using BET inhibitors; and (4) future directions, including novel BET bromodomain inhibitors, combinatorial approaches, and immunotherapy. It was concluded that standard chemotherapeutic approaches and first-generation BET bromodomain inhibitors, the latter complicated by a narrow therapeutic window, are only modestly effective in a minority of cases. Nonetheless, emerging second-generation targeted inhibitors, novel rational synergistic combinations, and the incorporation of immuno-oncology approaches hold promise to improve the prognosis of this disease.

The novel BET bromodomain inhibitor BI 894999 represses super-enhancer-associated transcription and synergizes with CDK9 inhibition in AML.

Bromodomain and extra-terminal (BET) protein inhibitors have been reported as treatment options for acute myeloid leukemia (AML) in preclinical models and are currently being evaluated in clinical trials. This work presents a novel potent and selective BET inhibitor (BI 894999), which has recently entered clinical trials (NCT02516553). In preclinical studies, this compound is highly active in AML cell lines, primary patient samples, and xenografts. HEXIM1 is described as an excellent pharmacodynamic biomarker for target engagement in tumors as well as in blood. Mechanistic studies show that BI 894999 targets super-enhancer-regulated oncogenes and other lineage-specific factors, which are involved in the maintenance of the disease state. BI 894999 is active as monotherapy in AML xenografts, and in addition leads to strongly enhanced antitumor effects in combination with CDK9 inhibitors. This treatment combination results in a marked decrease of global p-Ser2 RNA polymerase II levels and leads to rapid induction of apoptosis in vitro and in vivo. Together, these data provide a strong rationale for the clinical evaluation of BI 894999 in AML.

Synergistic activity of BET inhibitor BI 894999 with PLK inhibitor volasertib in AML in vitro and in vivo.

Interactions between a new potent Bromodomain and extraterminal domain (BET) inhibitor BI 894999 and the polo-like kinase (PLK) inhibitor volasertib were studied in acute myeloid leukemia cell lines in vitro and in vivo. We provide data for the distinct mechanisms of action of these two compounds with a potential utility in AML based on gene expression, cell cycle profile and modulation of PD biomarkers such as MYC and HEXIM1. In contrast to BI 894999, volasertib treatment neither affects MYC nor HEXIM1 expression, but augments and prolongs the decrease of MYC expression caused by BI 894999 treatment. In vitro combination of both compounds leads to a decrease in S-Phase and to increased apoptosis. In vitro scheduling experiments guided in vivo experiments in disseminated AML mouse models. Co-administration of BI 894999 and volasertib dramatically reduces tumor burden accompanied by long-term survival of tumor-bearing mice and eradication of AML cells in mouse bone marrow. Together, these preclinical findings provide evidence for the strong synergistic effect of BI 894999 and volasertib, warranting future clinical studies in patients with AML to investigate this paradigm.

Triple Angiokinase Inhibitor Nintedanib Directly Inhibits Tumor Cell Growth and Induces Tumor Shrinkage via Blocking Oncogenic Receptor Tyrosine Kinases.

The triple-angiokinase inhibitor nintedanib is an orally available, potent, and selective inhibitor of tumor angiogenesis by blocking the tyrosine kinase activities of vascular endothelial growth factor receptor (VEGFR) 1-3, platelet-derived growth factor receptor (PDGFR)-α and -ß, and fibroblast growth factor receptor (FGFR) 1-3. Nintedanib has received regulatory approval as second-line treatment of adenocarcinoma non-small cell lung cancer (NSCLC), in combination with docetaxel. In addition, nintedanib has been approved for the treatment of idiopathic lung fibrosis. Here we report the results from a broad kinase screen that identified additional kinases as targets for nintedanib in the low nanomolar range. Several of these kinases are known to be mutated or overexpressed and are involved in tumor development (discoidin domain receptor family, member 1 and 2, tropomyosin receptor kinase A (TRKA) and C, rearranged during transfection proto-oncogene [RET proto oncogene]), as well as in fibrotic diseases (e.g., DDRs). In tumor cell lines displaying molecular alterations in potential nintedanib targets, the inhibitor demonstrates direct antiproliferative effects: in the NSCLC cell line NCI-H1703 carrying a PDGFRα amplification (ampl.); the gastric cancer cell line KatoIII and the breast cancer cell line MFM223, both driven by a FGFR2 amplification; AN3CA (endometrial carcinoma) bearing a mutated FGFR2; the acute myeloid leukemia cell lines MOLM-13 and MV-4-11-B with FLT3 mutations; and the NSCLC adenocarcinoma LC-2/ad harboring a CCDC6-RET fusion. Potent kinase inhibition does not, however, strictly translate into antiproliferative activity, as demonstrated in the TRKA-dependent cell lines CUTO-3 and KM-12. Importantly, nintedanib treatment of NCI-H1703 tumor xenografts triggered effective tumor shrinkage, indicating a direct effect on the tumor cells in addition to the antiangiogenic effect on the tumor stroma. These findings will be instructive in guiding future genome-based clinical trials of nintedanib.

Pharmacological Profile of BI 847325, an Orally Bioavailable, ATP-Competitive Inhibitor of MEK and Aurora Kinases.

Although the MAPK pathway is frequently deregulated in cancer, inhibitors targeting RAF or MEK have so far shown clinical activity only in BRAF- and NRAS-mutant melanoma. Improvements in efficacy may be possible by combining inhibition of mitogenic signal transduction with inhibition of cell-cycle progression. We have studied the preclinical pharmacology of BI 847325, an ATP-competitive dual inhibitor of MEK and Aurora kinases. Potent inhibition of MEK1/2 and Aurora A/B kinases by BI 847325 was demonstrated in enzymatic and cellular assays. Equipotent effects were observed in BRAF-mutant cells, whereas in KRAS-mutant cells, MEK inhibition required higher concentrations than Aurora kinase inhibition. Daily oral administration of BI 847325 at 10 mg/kg showed efficacy in both BRAF- and KRAS-mutant xenograft models. Biomarker analysis suggested that this effect was primarily due to inhibition of MEK in BRAF-mutant models but of Aurora kinase in KRAS-mutant models. Inhibition of both MEK and Aurora kinase in KRAS-mutant tumors was observed when BI 847325 was administered once weekly at 70 mg/kg. Our studies indicate that BI 847325 is effective in in vitro and in vivo models of cancers with BRAF and KRAS mutation. These preclinical data are discussed in the light of the results of a recently completed clinical phase I trial assessing safety, tolerability, pharmacokinetics, and efficacy of BI 847325 in patients with cancer. Mol Cancer Ther; 15(10); 2388-98. ©2016 AACR.

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

Efficacy and mechanism of action of volasertib, a potent and selective inhibitor of Polo-like kinases, in preclinical models of acute myeloid leukemia.

Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immune-deficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.

Design, synthesis, and evaluation of indolinones as triple angiokinase inhibitors and the discovery of a highly specific 6-methoxycarbonyl-substituted indolinone (BIBF 1120).

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a new treatment modality in oncology. Preclinical findings suggest that blockade of additional pro-angiogenic kinases, such as fibroblast and platelet-derived growth factor receptors (FGFR and PDGFR), may improve the efficacy of pharmacological cancer treatment. Indolinones substituted in position 6 were identified as selective inhibitors of VEGF-, PDGF-, and FGF-receptor kinases. In particular, 6-methoxycarbonyl-substituted indolinones showed a highly favorable selectivity profile. Optimization identified potent inhibitors of VEGF-related endothelial cell proliferation with additional efficacy on pericyctes and smooth muscle cells. In contrast, no direct inhibition of tumor cell proliferation was observed. Compounds 2 (BIBF 1000) and 3 (BIBF 1120) are orally available and display encouraging efficacy in in vivo tumor models while being well tolerated. The triple angiokinase inhibitor 3 is currently in phase III clinical trials for the treatment of nonsmall cell lung cancer.

BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy.

Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional proangiogenic receptor tyrosine kinases: platelet-derived growth factor receptors (PDGFR) and fibroblast growth factor receptors (FGFR). BIBF 1120 is an indolinone derivative potently blocking VEGF receptor (VEGFR), PDGFR and FGFR kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120 inhibits mitogen-activated protein kinase and Akt signaling pathways in three cell types contributing to angiogenesis, endothelial cells, pericytes, and smooth muscle cells, resulting in inhibition of cell proliferation (EC(50), 10-80 nmol/L) and apoptosis. In all tumor models tested thus far, including human tumor xenografts growing in nude mice and a syngeneic rat tumor model, BIBF 1120 is highly active at well-tolerated doses (25-100 mg/kg daily p.o.), as measured by magnetic resonance imaging of tumor perfusion after 3 days, reducing vessel density and vessel integrity after 5 days, and inducing profound growth inhibition. A distinct pharmacodynamic feature of BIBF 1120 in cell culture is sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. Although BIBF 1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models. These distinctive pharmacokinetic and pharmacodynamic properties may help explain clinical observations with BIBF 1120, currently entering phase III clinical development.