Bmi1 Controls Tumor Development in an Ink4a/Arf-Independent Manner in a Mouse Model for Glioma

SummaryThe Polycomb group and oncogene Bmi1 is required for the proliferation of various differentiated cells and for the self-renewal of stem cells and leukemic cancer stem cells. Repression of the Ink4a/Arf locus is a well described mechanism through which Bmi1 can exert its proliferative effects. However, we now demonstrate in an orthotopic transplantation model for glioma, a type of cancer harboring cancer stem cells, that Bmi1 is also required for tumor development in an Ink4a/Arf-independent manner. Tumors derived from Bmi1;Ink4a/Arf doubly deficient astrocytes or neural stem cells have a later time of onset and different histological grading. Moreover, in the absence of Ink4a/Arf, Bmi1-deficient cells and tumors display changes in differentiation capacity

Extensive Metabolism and Hepatic Accumulation of Gemcitabine After Multiple Oral and Intravenous Administration in Mice

ABSTRACT: In a clinical study with oral gemcitabine (2,2-difluorodeoxycytidine, dFdC), we found that gemcitabine was hepatotoxic and extensively metabolized to 2,2-difluorodeoxyuridine (dFdU) after continuous oral dosing. The main metabolite dFdU had a long terminal half-life after oral administration. Our hypothesis was that dFdU and/or phosphorylated metabolites of gemcitabine accumulated in the liver after multiple oral dosing. In this study, mice were treated with oral or i.v. dFdC at a single dose (1qd؋1d) or at multiple doses once daily for 7 days (1qd؋7d) or seven times daily (7qd؋1d). Blood, liver, kidneys, and lungs were collected at several time points. Urine samples were collected after i.v. dFdC, and peripheral blood mononuclear cells were collected 7qd؋1d dosing of dFdC. The nucleosides dFdC and dFdU as well as the nucleotides gemcitabine monophosphate (dFdC-MP), diphosphate, and triphosphate (dFdC-TP) and dFdU monophosphate, diphosphate (dFdU-DP), and triphosphate (dFdU-TP) were simultaneously quantified by high-performance liquid chromatography with ultraviolet and radioisotope detection. We demonstrate that phosphorylated metabolites of both dFdC and dFdU are formed in mice, primarily consisting of dFdC-MP, dFdC-TP, and dFdU-TP. Multiple dosing of dFdC leads to substantial hepatic and renal accumulation of dFdC-TP and dFdU-TP, which have a more pronounced liver accumulation after oral than after i.v. dosing. The presence of dFdC-MP, dFdC-TP, and dFdU-TP in plasma and urine suggests efflux of these potentially toxic metabolites. Our results show that dFdU, dFdC-TP, and dFdU-TP accumulate in the liver after multiple dosing of dFdC in mice and might be associated with hepatotoxicity of oral dFdC in patients. Gemcitabine (2Ј,2Ј-difluorodeoxycytidine, dFdC), a pyrimidine nucleoside anticancer drug, is used in the treatment of patients with a variety of solid tumors Alternatively, dFdC is deaminated to 2Ј,2Ј-difluorodeoxyuridine (dFdU) by cytidine deaminase (CDA), which is highly expressed in human liver and mice kidney In a clinical study, dFdC was orally administered in continuous dosing regimens at low dose levels in patients with advanced solid tumors . The exposure to dFdC was low because of extensive first-pass metabolism to dFdU. Additionally, we found that the triphosphate form of dFdU (dFdU-TP) was formed at high exposure levels in peripheral blood mononuclear cells (PBMCs). One patient treated with 8 mg of oral dFdC once daily for 14 days of a 21-day cycle developed lethal hepatic toxicity during the second cycle. Pathological examination revealed severe drug-induced liver necrosis. Pharmacokinetic analysis demonstrated that dFdU has a long terminal half-life (t 1/2 ) (ϳ89 h) and appeared to accumulate in the liver of patients. Based on these findings, we hypothesized that continuous daily oral dosing of dFdC results in liver accumulation of dFdU and/or phosphorylated metabolites in patients, possibly associated with the hepatotoxicity of dFdC. We recently found that dFdU is Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.021048. ABBREVIATIONS: dFdC, 2Ј,2Ј-difluorodeoxycytidine (gemcitabine); dCK, deoxycytidine kinase; dFdC-MP, gemcitabine monophosphate; dFdC-TP, gemcitabine triphosphate; dFdC-DP, gemcitabine diphosphate; dFdU, 2Ј,2Ј-difluorodeoxyuridine; CDA, cytidine deaminase; dFdU-TP, dFdU triphosphate; PBMC, peripheral blood mononuclear cell; hCNT1, human concentrative nucleoside transporter type 1; PK, pharmacokinetics; 1qdϫ1d, single dose on day 1; 1qdϫ7d, once daily dosing for 7 days; 7qdϫ1d, seven times daily dosing for 1 day; dFdU-MP, dFdU monophosphate; dFdU-DP, dFdU diphosphate; THU, tetrahydrouridine; AP, alkaline phosphatase; HPLC, high-performance liquid chromatography; AUC, area under the curve

Multiple low dose therapy as an effective strategy to treat EGFR inhibitor-resistant NSCLC tumours

Resistance to targeted cancer drugs is thought to result from selective pressure exerted by a high drug dose. Partial inhibition of multiple components in the same oncogenic signalling pathway may add up to complete pathway inhibition, while decreasing the selective pressure on each component to acquire a resistance mutation. We report here testing of this Multiple Low Dose (MLD) therapy model in EGFR mutant NSCLC. We show that as little as 20% of the individual effective drug doses is sufficient to completely block MAPK signalling and proliferation when used in 3D (RAF + MEK + ERK) or 4D (EGFR + RAF + MEK + ERK) inhibidor combinations. Importantly, EGFR mutant NSCLC cells treated with MLD therapy do not develop resistance. Using several animal models, we find durable responses to MLD therapy without associated toxicity. Our data support the notion that MLD therapy could deliver clinical benefit, even for those having acquired resistance to third generation EGFR inhibidor therapy.This work was supported by a grant from the Dutch Cancer Society through the Oncode Institute. Al.V. was supported by the Fondo de Investigaciones Sanitarias, FIS (PI16-01898, and by the Spanish Association Against Cancer, AECC (CGB14142035THOM) and Ideas Semilla project (IDEAS098VILL-IDEAS16) and Generalitat de Catalunya (2014SGR364). L.F. received a European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie, grant agreement number 799850. E.N. was funded by Instituto Carlos III through the project PI18/00920. We thank CERCA Program/Generalitat de Catalunya for their institutional support and grant 2017SGR448

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity in vitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.This work was funded by the American Association for Cancer Research, Lustgarten Foundation, and Stand Up to Cancer as a Pancreatic Cancer Collective New Therapies Challenge grant (grant no. SU2C-AACR-PCC-01-18)

Tryptophan depletion results in tryptophan-to-phenylalanine substitutants

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1–4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides ‘substitutants’ to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity

Concerns about anti-angiogenic treatment in patients with glioblastoma multiforme

BACKGROUND: The relevance of angiogenesis inhibition in the treatment of glioblastoma multiforme (GBM) should be considered in the unique context of malignant brain tumours. Although patients benefit greatly from reduced cerebral oedema and intracranial pressure, this important clinical improvement on its own may not be considered as an anti-tumour effect. DISCUSSION: GBM can be roughly separated into an angiogenic component, and an invasive or migratory component. Although this latter component seems inert to anti-angiogenic therapy, it is of major importance for disease progression and survival. We reviewed all relevant literature. Published data support that clinical symptoms are tempered by anti-angiogenic treatment, but that tumour invasion continues. Unfortunately, current imaging modalities are affected by anti-angiogenic treatment too, making it even harder to define tumour margins. To illustrate this we present MRI, biopsy and autopsy specimens from bevacizumab-treated patients. Moreover, while treatment of other tumour types may be improved by combining chemotherapy with anti-angiogenic drugs, inhibiting angiogenesis in GBM may antagonise the efficacy of chemotherapeutic drugs by normalising the blood-brain barrier function. SUMMARY: Although angiogenesis inhibition is of considerable value for symptom reduction in GBM patients, lack of proof of a true anti-tumour effect raises concerns about the place of this type of therapy in the treatment of GBM