Abstract The well-established capability of tumors to resist single agent chemotherapies underscores the need to identify novel synergistic drug combinations to treat patients with cancer. Here we use an innovative arrayed needle technology to provide rapid in vivo validation of findings from a genome scale RNAi screen and reveal the potential of an unexpected drug combination to treat patients with melanoma. Our efforts focused on the β-catenin pathway. The β-catenin pathway impacts cancer progression in a context dependent manner. In some malignancies such as colon cancer, increased β-catenin activity drives oncogenesis. In contrast activation of β-catenin has been associated with decreased tumor proliferation and improved prognosis in patients with melanoma. A screen with an arrayed library of 14,000 lentiviral shRNA vectors revealed that silencing of dihydrofolate reductase, the target of the classic chemotherapeutic methotrexate, led to synergistic activation of the β-catenin pathway when combined with an inhibitor of GSK3. We therefore explored whether combination therapy, consisting of a GSK3 inhibitor and methotrexate could represent a potential treatment for melanoma patients. Two distinct small molecule inhibitors of GSK3, 6-bromoindirubin-3αoxime (BIO) and Chiron 99021, demonstrated synergy with methotrexate to activate β-catenin signaling in the melanoma cell line A375. To rapidly assess whether the observed synergy translates to the context of a living tumor, precision multiplexed microinjection using Presage technology was performed on mice harboring flank melanoma tumors. Discreet positions on each tumor were injected with either vehicle control, methotexate as a single agent, Chiron 99021 as a single agent, or the combination of the two agents. Analysis of the activation of beta-catenin and histology for markers of cellular proliferation, death, and differentiation allowed us to rapidly evaluate the efficacy of this drug combination. This rapid in vivo validation approach sets the stage for full preclinical evaluation of combined methotrexate and GSK3 inhibitor therapy using advanced co-clinical models of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5116. doi:1538-7445.AM2012-5116
We are developing an indicator cell assay platform (iCAP) for blood-based diagnosis of early stages of Alzheimer's disease (AD). This assay uses standardized, commercially-sourced neurons from a single normal individual as biosensors that detect and respond to AD signals in plasma. The diagnostic readout is the expression level of a small number of selected genes in the indicator cells. We developed an iCAP for AD diagnosis and trained and validated iCAP classifiers with plasma samples from three separate sources. Case samples were from subjects with presymptomatic AD or mild cognitive impairment (MCI) due to AD, and control samples were from cognitively normal individuals or patients with non-AD dementia. Classes were defined by CDR scores and Abeta42 levels detected by imaging or CSF analysis. Classifiers were tested by blind independent validation at two stages of data collection. With training and validation sets of 101 and 41 samples, respectively, the assay achieved 69% sensitivity and 91% specificity in distinguishing early stages of AD from normal controls (AUC of ROC = 0.76; ANOVA p-value 0.0065). With training and validation sets of 186 and 50, respectively, the assay distinguished subjects at early stages of AD from normal controls and those with non-AD dementia with AUC of ROC = 0.68 (p-value 0.023; FDR 0.14). Both classifiers used samples from 3 separate sources. This work establishes a paradigm for using cultured cells as biosensors for blood-based diagnosis of AD before clinical onset.
Abstract Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis by preventing excessive inflammation in normal tissues. In cancer, Tregs hamper antitumor immunosurveillance and may be a resistance mechanism to anti-PD-1 therapies. In preclinical cancer models, Treg depletion enhances antitumor cytotoxic immune responses, but systemic and persistent Treg removal can elicit immunopathology. Therapeutic strategies that selectively deplete highly suppressive activated intratumoral Tregs may avoid immune-related toxicities and amplify the activity of antitumor CD8 T cells during PD-1 blockade. CD30 is a member of the TNF receptor superfamily and is expressed by a subset of activated lymphocytes and various lymphomas. Here, transcriptomics and flow cytometry data from tumor and peripherally derived Tregs demonstrated CD30 is enriched on intratumoral Tregs. Among peripheral Tregs, CD30 expression was associated with an activated effector phenotype (FoxP3hiCD45RA-, fraction II). Further, treatment of dissociated NSCLC tumor spheroids with anti-PD-1 resulted in upregulated CD30 expression by Tregs but not intratumoral CD4 or CD8 T cells, which is consistent with Treg reactivation and supports CD30 as an activated Treg target. In vitro, CD30 expression on Tregs was dependent on signaling through the TCR and was significantly amplified by IL-2 and IL-15, suggesting CD30 may identify a subpopulation of activated antigen-specific Tregs. Brentuximab vedotin (BV) is an antibody-drug conjugate approved for clinical use in multiple types of lymphomas including cHL and PTCL. BV consists of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE). The activity of BV in lymphomas is thought to primarily result from tumor-directed intracellular MMAE release leading to apoptosis, though multiple immunomodulatory effects of BV have also been suggested. We have demonstrated through in vitro and in vivo preclinical model systems of human T cell activation that BV can selectively deplete CD30-expressing Tregs, amplifying cytotoxic CD8 T cell expansion. These and Treg CD30 expression data motivated the clinical exploration of BV plus pembrolizumab in metastatic PD-1 refractory melanoma and NSCLC (NCT04609566). Consistent with preclinical data, preliminary biomarker analysis from these studies showed a small but significant decrease in Tregs (CD4+CD25+CD127low/-) in peripheral blood after treatment with BV plus pembrolizumab. Further, limited on-treatment biopsies showed a trend toward increased intratumoral CD8:Treg (FoxP3) ratio in some patients receiving combination therapy. These data support the ongoing evaluation of BV in combination with PD-1 inhibitors in solid tumors and a potential additional mechanism of action for BV. Citation Format: Ryan A. Heiser, Bryan M. Grogan, Reice D. James, Michelle L. Ulrich, Jason D. Berndt, Brian P. O'Connor, Shyra J. Gardai, Hailing Lu, Scott M. Knowles. CD30 is a marker of activated effector regulatory T cells in solid tumors providing clinical rationale for the combination of brentuximab vedotin and PD-1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3253.
Cell Biology
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is important during the development of the nervous system. ALK can also be aberrantly activated in certain types of cancers, including neuroblastoma and lung adenocarcinoma. Until now, ALK was an “orphan” receptor because its ligand was not known. Now, Murray et al. show that heparin binds directly to ALK and stimulates its activity (see the Focus by Lemke). An antibody that blocked the binding prevented heparin from activating this receptor tyrosine kinase, providing a potential avenue for therapeutic intervention.
Sci. Signal. 8 , ra6; see also fs2 (2015).
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