In multiple sclerosis (MS), B cell-depleting therapy using monoclonal anti-CD20 Abs, including rituximab (RTX) and ocrelizumab, effectively reduces disease activity. Based on indirect evidence, it is ...generally believed that elimination of the Ag-presenting capabilities and Ag nonspecific immune functions of B cells underlie the therapeutic efficacy. However, a small subset of T lymphocytes (T cells) was shown to also express CD20, but controversy prevails surrounding the true existence of this T cell subpopulation. Using single-cell imaging flow cytometry and expression profiling of sorted lymphocyte subsets, we unequivocally demonstrate the existence of CD3(+)CD20(dim) T cells. We show that in MS patients, increased levels of CD3(+)CD20(dim) T cells are effectively depleted by RTX. The pathological relevance of this T cell subset in MS remains to be determined. However, given their potential proinflammatory functionality, depletion of CD20-expressing T cells may also contribute to the therapeutic effect of RTX and other mAbs targeting CD20.
Necroptosis is a lytic form of cell death that is mediated by the kinase RIPK3 and the pseudokinase MLKL when caspase-8 is inhibited downstream of death receptors, toll-like receptor 3 (TLR3), TLR4, ...and the intracellular Z-form nucleic acid sensor ZBP1. Oligomerization and activation of RIPK3 is driven by interactions with the kinase RIPK1, the TLR adaptor TRIF, or ZBP1. In this study, we use immunohistochemistry (IHC) and in situ hybridization (ISH) assays to generate a tissue atlas characterizing RIPK1, RIPK3, Mlkl, and ZBP1 expression in mouse tissues. RIPK1, RIPK3, and Mlkl were co-expressed in most immune cell populations, endothelial cells, and many barrier epithelia. ZBP1 was expressed in many immune populations, but had more variable expression in epithelia compared to RIPK1, RIPK3, and Mlkl. Intriguingly, expression of ZBP1 was elevated in Casp8
Tnfr1
embryos prior to their succumbing to aberrant necroptosis around embryonic day 15 (E15). ZBP1 contributed to this embryonic lethality because rare Casp8
Tnfr1
Zbp1
mice survived until after birth. Necroptosis mediated by TRIF contributed to the demise of Casp8
Tnfr1
Zbp1
pups in the perinatal period. Of note, Casp8
Tnfr1
Trif
Zbp1
mice exhibited autoinflammation and morbidity, typically within 5-7 weeks of being born, which is not seen in Casp8
Ripk1
Trif
Zbp1
, Casp8
Ripk3
, or Casp8
Mlkl
mice. Therefore, after birth, loss of caspase-8 probably unleashes RIPK1-dependent necroptosis driven by death receptors other than TNFR1.
Abstract
Lung adenocarcinomas comprise the largest fraction of non-small cell lung cancer, which is the leading cause of cancer deaths. 75% of adenocarcinomas lack targeted therapies due to scarcity ...of druggable drivers. We leveraged transcriptional data from >800 early-stage and advanced patients to classify tumors based on signaling similarities and discover subgroups within this unmet patient population. The subtypes capture heterogeneity even amongst tumors lacking known oncogenic drivers. Paired multi-regional intratumoral biopsies demonstrate unified subtypes despite divergently evolved pro-oncogenic mutations, indicating subtype stability during selective pressure. We identified differential subtype response to MEK inhibition across multiple preclinical model systems and a clinical trial, supporting prognostic utility of transcriptional subtyping. Differential subtype dependency on MEK signaling reproduced in a mouse model of KRAS-mutant lung adenocarcinoma, where a MEK-dependent adenocarcinoma subtype is driven by a SOX2 cellular state. Our findings support forward translational relevance of transcriptional subtypes and reveal that naturally evolved yet ectopic expression of a pioneer transcription factor may modulate tumor subtype and response.
Citation Format: Jonathan E. Cooper, Anneleen Daemen, Dorothee Nickles, Szymon Myrta, Oded Foreman, Jeff Eastham, Melissa R. Junttila, Heinrich Jasper. SOX2 delineates a mouse lung adenocarcinoma subtype vulnerable to targeted therapy abstract. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-099.
Major advances in the genetics of multiple sclerosis (MS) have been reported in 2011. These include studies in gene mapping, functional characterization of previously associated genes, and the ...relationship between genes and the environment. While particularly true for gene discovery, each of these efforts requires substantial statistics and computational resources for adequate analysis. This review describes the major advances in the genetics of MS with a slight emphasis on data handling and analysis.
Articles discussed include a new genome-wide association study (GWAS) with almost 10 000 cases (a collaboration between the Wellcome Trust and the international MS Genetics Consortium) that identified new susceptibility loci, taking the total number of risk alleles to more than 50. An article describing the use of next-generation sequencing to identify a rare mutation in CYP27B1 in a MS family is also discussed. Moreover, a summary of recent reports describing functional studies of MS-associated genes as well as the latest research on the interactions between genes and the environment is provided.
This review provides a concise summary of the most relevant studies in the genetics of MS in the past year. We raise awareness about analytical resources to successfully analyze the massive datasets characteristic of today's genetic studies.
T cell-retargeting therapies have transformed the therapeutic landscape for hematologic diseases. T cell-dependent bispecific antibodies (TDB) function as conditional agonists that induce a ...polyclonal T-cell response, resulting in target cell destruction and cytokine release. The relationship between this response and its effects on surrounding innate immune populations has not been fully explored. Here we show that treatment with mosunetuzumab in patients results in natural killer (NK) cell activation in the peripheral blood. We modeled this phenomenon in vitro and found that TDB-mediated killing activated NK cells, increasing NK function and antibody-dependent cellular cytotoxicity (ADCC), and enhanced the capability of macrophages to perform antibody-dependent cellular phagocytosis (ADCP). This enhancement was triggered by cytokines released through TDB treatment, with IL2 and IFNγ being major drivers for increased ADCC and ADCP, respectively. Surprisingly, cytolytic ability could be further augmented through neutralization of IL10 for NK cells and TNFα for macrophages. Finally, we showed that TDB treatment enhanced the efficacy of Fc-driven killing to an orthogonal solid tumor target in vivo. These results provide rationale for novel antibody therapy combinations that take advantage of both adaptive and innate immune responses.
Abstract
Introduction: Inflammatory breast cancer (IBC) is a clinical diagnosis that spans all breast cancer subtypes. The clinical course of IBC is associated with poorer outcomes than molecular ...subtype-matched non-IBC and represents an unmet need in breast cancer therapy. IBC is characterized by invasive dermal lymphatic tumor emboli (DLTE) resulting in erythema and edema of the breast. This suggests a common molecular theme, which if identified and targeted, could reduce mortality. Although several genomic and transcriptomic studies of IBC have been performed, no definitive genomic drivers have been identified. We hypothesized that the genomic features of IBC remain undiscovered because only the primary tumor has been analyzed, rather than the population of tumor cells responsible for the phenotype (i.e. DLTE). However, analysis of DLTE is challenging due to lack of effective technologies to purify these cells from the more abundant stroma.
Methods: We utilized the Millisect automated dissection (AD) technology, which can selectively recover tissue from targeted areas as small as 200µ2 on standard FFPE sections, on matched skin with DLTE and primary tumor slides from 7 post-treatment IBC mastectomy specimens. We performed RNA sequencing (RNAseq) on AD-enriched primary tumor and DLTE, on full sections from the same samples (including stroma and tumor) and on tissue remaining post-AD (containing residual stroma after tumor cell extraction).
Results: RNAseq analysis in AD-enriched primary tumor and DLTEs identified unique transcriptional patterns upregulated in DLTE indicative of lymphatic trafficking (e.g. CCL21), immunosuppression (e.g. S100A9/calprotectin), and myofibroblastomic differentiation (e.g. CD34, desmin, alpha-smooth muscle actin). Gene set enrichment corroborated these inferences, demonstrating DLTE enrichment of gene sets involved in chemokine/trafficking, tumor stemness, and smooth muscle contraction/migration. These gene sets were not apparent in samples extracted from the full sections.
Conclusions: Automated dissection technology enables specific investigation on the limited epithelial material that comprises inflammatory breast cancer involving dermal lymphatics. Further investigation, with cohort expansion to increase sample size and statistical power, will focus on pre-treatment skin biopsies with DTLE and matched breast tumor tissue. We expect this will yield novel insights into the biology and treatment of this unique phenotype.
Citation Format: Jennifer M Giltnane, Justin M Balko, Nickles Dorothee, Sarajane Nghiem, Anneleen Daemen, Emmanuel Naouri, Amy A Lo, Beth T Harrison, Emily J Schlosnagle, Charles Havnar, Carmina Espiritu, Daniel G Stover, Beth A Overmoyer. Automated tissue dissection of dermal lymphatic emboli in inflammatory breast cancer enhances accuracy of transcriptional analysis abstract. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-15-02.
Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain ...containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient
antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.
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326
Background: TCGA molecular sybtyping is established in metastatic urothelial bladder cancer (UBC). It remains unclear if it is of prognostic importance with chemotherapy in ...metastatic UBC. Data into immune therapy suggests TCGA subtyping is predictive to response. Methods: Archival formalin-fixed paraffin embedded (FFPE) tissue were analysed from 170 patients that had consented into a clinical trial (NCT00949455) after first line chemotherapy for metastatic disease. Gene expression levels were quantified by NanoString technology. Molecular subtypes were assigned according to The Cancer Genome Atlas (TCGA) subtypes. Clinical information was available for all patients and analysis on individual genes were explored. Results: 170 patients were analysed, 75% male. 107 (64%) patients received cisplatin based first-line chemotherapy, with 36% receiving carboplatin based regimen. Median overall survival (OS) was 15.73 months 95% CI: 13.87-17.58, and median progression free survival (PFS) was 10.71 months 95% CI: 8.97-12.45. Samples were initially clustered into luminal (n=109) and basal (n=61) subtypes. Response to first line chemotherapy occurred in all subtypes but was shown to be significantly higher in the luminal subtype versus the basal subtype 58% vs 20%, p=0.01. PFS was superior in luminal subtypes 11.8 months vs 8.9 months, p=0.005. Exploratory analysis showed that luminal II subtype had the best outcome for OS 20.3 months, p=0.03 compared to the other subtypes. Outcomes with other genes including immune markers were explored. TCGA outcomes can be summarised in the table. Conclusions: In metastatic urothelial cancer, TCGA subclassifying influences outcome of patients post chemotherapy. Table: see text
Tumor necrosis factor α (TNF‐α) signaling pathways play important roles during tumorigenesis and inflammation. Ubiquitin‐dependent processes are central to the regulation of TNF‐α and nuclear factor ...κB (NF‐κB) signaling. We performed a targeted siRNA screen for ubiquitin‐specific proteases (USPs) and identified USP2 as a modulator of TNF‐α‐induced NF‐κB signaling. We showed that USP2 is required for the phosphorylation of IκB, nuclear translocation of NF‐κB and expression of NF‐κB‐dependent target genes and IL‐8 secretion. Our study also provides evidence for isoform‐specific functions of USP2. The immunohistochemical analysis of breast carcinomas revealed that USP2 expression is frequently downregulated. Together, our results implicate USP2 as a novel positive regulator of TNF‐α‐induced NF‐κB signaling and show that its expression is altered in tumor cells.
Abstract
Reversible EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib offer significant clinical benefit to patients with EGFR mutation positive non-small cell lung cancer ...(NSCLC) compared to chemotherapy alone, but high rates of resistance especially at EGFR T790M (50-60% of resistant cases) underscore the need for better targeted treatments for NSCLC patients. Third generation irreversible TKIs–AZD9291 and CO-1686–were developed for efficacy in the T790M setting. However, resistance to the new class of EGFR TKIs is inevitable, prompting us to investigate the relationship between erlotinib and AZD9291 resistance mechanisms and explore novel ones. NSCLC cell lines harboring activating EGFR mutations with (NCI-H1975) or without T790M (HCC4006, HCC827, and PC-9) were exposed to erlotinib, dacomitnib, or AZD9291 at IC 75 or greater for three months to generate resistance. We observed cross-resistance to other EGFR TKIs in nearly all resistant lines, suggesting that T790M was not the main driver of resistance in the erlotinib-treated lines and that a common mechanism might account for resistance in each cell line set. An epithelial to mesenchymal transition (EMT) was observed and a corresponding increase in vimentin staining was seen in resistant lines, consistent with cross-resistance. Previous studies showed that MET amplification caused erlotinib resistance in HCC827; our data suggest MET amplification also accounted for the AZD9291 and dacomitnib resistance in HCC827. In AZD9291-resistant HCC4006 and H1975 lines, PTEN levels were substantially reduced compared to the parental line, with a corresponding increase in PIK3 pathway markers. Whole exome sequencing data support homozygous deletion of PTEN loss as a mechanism of acquired resistance to AZD9291 in NCI-H1975. The PTEN null line NCI-H1650 was found to be resistant to all three TKIs without long-term drug exposure, implicating PTEN loss as a mechanism of de novo resistance. An analysis of EGFR mutation status, PTEN status, MET expression, and the kinetics of acquired resistance will be presented and clinical implications will be discussed.
Citation Format: Sarah M. Paul, Dorothee Nickles, Xioafen Ye, Robert L. Yauch, David S. Shames. Cross-resistance to 1st, 2nd, and 3rd generation EGFR tyrosine kinase inhibitors in vitro is characterized by MET amplification and PTEN loss. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3602. doi:10.1158/1538-7445.AM2015-3602
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