The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and ...characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH.
A distinctive feature of BRCA1-linked breast cancers is that they typically do not express estrogen receptor-alpha (ER(alpha)). Previous investigation suggests that methylation of CpGs within the ...ER(alpha) promoter mediates repression of gene expression in some ER(alpha)-negative breast cancers. To determine if methylation of CpGs within the ER(alpha) promoter is associated with BRCA1-linked breast cancers, we evaluated methylation in exon 1 of the ER(alpha) gene in 40 ER(alpha)-negative breast cancers, 20 of which were non BRCA1-linked and 20 BRCA1-linked. CpG methylation was evaluated by either methylation-sensitive restriction digest (HpaII), methylation-sensitive PCR (MSP), or direct sequencing of bisulfite-treated genomic DNA. Results from HpaII digests and MSP documented a high degree of methylation, the MSP data showing slightly higher methylation in the BRCA1-linked group. CpGs analysed by direct sequencing showed an overall average methylation of 25% among non BRCA1-linked cancers and 40% among BRCA1-linked cancers (P=0.0031). The most notable difference was found at five particular CpGs, each of which exhibited a greater than twofold increase in methylation in the BRCA1-linked group compared to the non BRCA1-linked group (P<0.03 for each CpG). Methylation of certain critical CpGs may represent an important factor in transcriptional repression of the ER(alpha) gene in BRCA1-linked breast cancers.
Pim kinases have been shown to play a key role as downstream effectors of growth factor signaling in hematological malignancies. We have previously described AZD1208, a novel, orally bioavailable, ...highly selective pan Pim kinase inhibitor, undergoing clinical testing in AML. Specifically, we had demonstrated the anti-proliferative activity of AZD1208 in models of AML and had shown that AZD1208 treatment of AML cell lines results in the dose dependent reduction of pBAD at serine 112 and p4EBP1 at serine 65 as well as pp70S6K at threonine 389 and pS6 at serine 235/236, associated with global effects on protein translation (Keeton et al. Blood 2014).
Since AZD1208 is about 10-15 fold more potent against Pim-1 than Pim-2 in both enzymatic and cellular assays, we sought to assess the dependency of each of these markers on Pim-1 and/or Pim-2 for phosphorylation using a Pim-1/3 selective inhibitor (Pim-1/3) that lacks Pim-2 inhibitory activity. In vitro studies demonstrate that while the modulation of pBAD does not require inhibition of Pim-2, the regulation of 4EBP1 as well as p70S6K and S6 are dependent, at least in part, on Pim-2 activity. As these proteins are canonically regulated by mTORC1, these data are consistent with Pim-2 acting upstream of this complex; perhaps through phosphorylation of TSC2 as reported for multiple myeloma cell lines (Lu et al. Blood 2013).
These observations are also supported by in vivo data. Analysis of pBAD and p4EBP1 levels in Molm16 xenografts show that maximal inhibition of pBAD (70-80% inhibition) is achieved at AZD1208 plasma concentration of about 1000 ng/ml, consistent with the estimated cellular IC90 of pBAD inhibition in vitro. However, maximal inhibition of p4EBP1 is achieved at concentrations 3-5 fold higher. This observation is consistent with the differential potency of AZD1208 against Pim-1 and Pim-2, and with a requirement for Pim-2 inhibition for maximal p4EBP1 inhibition in this AML model.
pBAD and p4EBP1 were selected as the pharmacodynamic endpoints for evaluating clinical target inhibition in patient samples. Bone marrow and peripheral blood samples were collected pre- and post-dose from relapsed, refractory AML patients enrolled in the AZD1208 Phase I dose escalation. Analysis of pBAD inhibition on day 1 of dosing shows at least 60-70% inhibition across all of the dosing cohorts. With clinical exposures on day 1 approaching 1000 ng/ml at the first dose level and exceeding 1000 ng/ml as the dose increases, the extent of pBAD inhibition seen in patients appears to be consistent with Pim-1 inhibition, as seen in preclinical models. Furthermore, and also similar to our preclinical observations, maximal inhibition of p4EBP1 in patients is achieved only at higher exposures. These data strengthen the hypothesis that BAD phosphorylation is primarily dependent on Pim-1, whereas suppression of Pim-2 activity is required for maximal inhibition of p4EBP1 in AML cells.
In summary, Pim inhibition in AML cell line models and in patients treated with AZD1208 results in the inhibition of the downstream targets of Pim signaling, pBAD and p4EBP1. Invitro and in vivo, the inhibition of pBAD is consistent with inhibition of Pim-1 while inhibition of p4EBP1 indicates a requirement for Pim-2 inhibition as well. These observations are validated in patients and provide further evidence for the relevance of these biomarkers as a measure of Pim signaling in AML.
McEachern:AstraZeneca: Employment, Equity Ownership. O’Connor:AstraZeneca: Employment, Equity Ownership. DuPont:AstraZeneca: Employment, Equity Ownership. Gibbons:AstraZeneca: Employment, Equity Ownership. Pablo:AstraZeneca: Employment, Equity Ownership. Vishwanathan:AstraZeneca: Employment, Equity Ownership. McCoon:AstraZeneca: Employment, Equity Ownership. Cortes:AstraZeneca: Research Funding. Neumann:AstraZeneca: Employment, Equity Ownership. Keating:AstraZeneca: Employment, Equity Ownership. Pease:AstraZeneca: Employment, Equity Ownership. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Barrett:AstraZeneca: Employment, Equity Ownership.
Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby ...limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.
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•TIM-3 blockade promotes endocytosis of extracellular DNA by dendritic cells•DNA uptake and CXCL9 expression by dendritic cells is HMGB1 dependent•Galectin-9 regulates TIM-3 cell surface clustering and inhibitory function•Antitumor efficacy of TIM-3 mAb and paclitaxel is dependent upon cGAS and STING
Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. de Mingo Pulido et al. provide insight into the underlying mechanisms by revealing that TIM-3 suppresses HMGB1-dependent endocytosis of extracellular DNA and the subsequent activation of the cGAS-STING pathway in intra-tumoral dendritic cells.
To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non-small cell lung cancer (NSCLC).
Using ...multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment.
PD-1, LAG-3, and TIM-3 were detected in tumor-infiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progression-free survival.
PD-1, LAG-3, and TIM-3 have distinct tissue/cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.
Upregulation of Pim kinases is observed in several types of leukemias and lymphomas. Pim-1, -2, and -3 promote cell proliferation and survival downstream of cytokine and growth factor signaling ...pathways. AZD1208 is a potent, highly selective, and orally available Pim kinase inhibitor that effectively inhibits all three isoforms at <5 nM or <150 nM in enzyme and cell assays, respectively. AZD1208 inhibited the growth of 5 of 14 acute myeloid leukemia (AML) cell lines tested, and sensitivity correlates with Pim-1 expression and STAT5 activation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells, accompanied by a dose-dependent reduction in phosphorylation of Bcl-2 antagonist of cell death, 4EBP1, p70S6K, and S6, as well as increases in cleaved caspase 3 and p27. Inhibition of p4EBP1 and p-p70S6K and suppression of translation are the most representative effects of Pim inhibition in sensitive AML cell lines. AZD1208 inhibits the growth of MOLM-16 and KG-1a xenograft tumors in vivo with a clear pharmacodynamic-pharmacokinetic relationship. AZD1208 also potently inhibits colony growth and Pim signaling substrates in primary AML cells from bone marrow that are Flt3 wild-type or Flt3 internal tandem duplication mutant. These results underscore the therapeutic potential of Pim kinase inhibition for the treatment of AML.
•AZD1208 is a selective pan-Pim kinase inhibitor with efficacy in AML cells, xenografts, and Flt3-internal tandem duplication or Flt3 wild-type patient samples.•AML cell growth inhibition is associated with suppression of p70S6K, 4EBP1 phosphorylation, and messenger RNA translation.
Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine ...receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using short hairpin RNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.
Patients with repaired Tetralogy of Fallot (rTOF) experience a high burden of long-term morbidity, particularly arrhythmias. Cardiovascular magnetic resonance (CMR) is routinely used to assess ...ventricular characteristics but the relationship between CMR diastolic function and arrhythmia has not been evaluated. We hypothesized in rTOF, left ventricular (LV) diastolic dysfunction on CMR would correlate with arrhythmias and mortality.
Adolescents and adults with rTOF who underwent CMR were compared to healthy controls (n = 58). Standard ventricular parameters were assessed and manual planimetry was performed to generate filling curves and indices of diastolic function. Chart review was performed to collect outcomes. Univariate and multivariable logistic regression was performed to identify outcome associations.
One-hundred sixty-seven subjects with rTOF (mean age 32 years) and 58 healthy control subjects underwent CMR. Patients with rTOF had decreased LV volumes and increased right ventricular (RV) volumes, lower RV ejection fraction (RVEF), lower peak ejection rate (PER), peak filling rate (PFR) and PFR indexed to end-diastolic volume (PFR/EDV) compared to healthy controls. Eighty-three subjects with rTOF had arrhythmia (63 atrial, 47 ventricular) and 11 died. Left atrial (LA) volumes, time to peak filling rate (tPFR), and PFR/EDV were associated with arrhythmia on univariate analysis. PER/EDV was associated with ventricular (Odds ratio, OR 0.43 0.24–0.80, p = 0.007) and total arrhythmia (OR 0.56 0.37–0.92, p = 0.021) burden. A multivariable predictive model including diastolic covariates showed improved prediction for arrhythmia compared to clinical and conventional CMR measures (area under curve (AUC) 0.749 v. 0.685 for overall arrhythmia). PFR/EDV was decreased and tPFR was increased in rTOF subjects with mortality as compared to those without mortality.
Subjects with rTOF have abnormal LV diastolic function compared to healthy controls. Indices of LV diastolic function were associated with arrhythmia and mortality. CMR diastolic indices may be helpful in risk stratification for arrhythmia.
Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of ...patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.
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