Novel microenvironment-based classification of intrahepatic cholangiocarcinoma with therapeutic implications.

OBJECTIVE: The diversity of the tumour microenvironment (TME) of intrahepatic cholangiocarcinoma (iCCA) has not been comprehensively assessed. We aimed to generate a novel molecular iCCA classifier that incorporates elements of the stroma, tumour and immune microenvironment ('STIM' classification). DESIGN: We applied virtual deconvolution to transcriptomic data from ~900 iCCAs, enabling us to devise a novel classification by selecting for the most relevant TME components. Murine models were generated through hydrodynamic tail vein injection and compared with the human disease. RESULTS: iCCA is composed of five robust STIM classes encompassing both inflamed (35%) and non-inflamed profiles (65%). The inflamed classes, named immune classical (~10%) and inflammatory stroma (~25%), differ in oncogenic pathways and extent of desmoplasia, with the inflammatory stroma showing T cell exhaustion, abundant stroma and KRAS mutations (p<0.001). Analysis of cell-cell interactions highlights cancer-associated fibroblast subtypes as potential mediators of immune evasion. Among the non-inflamed classes, the desert-like class (~20%) harbours the lowest immune infiltration with abundant regulatory T cells (p<0.001), whereas the hepatic stem-like class (~35%) is enriched in 'M2-like' macrophages, mutations in IDH1/2 and BAP1, and FGFR2 fusions. The remaining class (tumour classical: ~10%) is defined by cell cycle pathways and poor prognosis. Comparative analysis unveils high similarity between a KRAS/p19 murine model and the inflammatory stroma class (p=0.02). The KRAS-SOS inhibitor, BI3406, sensitises a KRAS-mutant iCCA murine model to anti-PD1 therapy. CONCLUSIONS: We describe a comprehensive TME-based stratification of iCCA. Cross-species analysis establishes murine models that align closely to human iCCA for the preclinical testing of combination strategies.

Intrinsic Gata4 expression sensitizes the aortic root to dilation in a Loeys-Dietz syndrome mouse model.

Loeys-Dietz syndrome (LDS) is an aneurysm disorder caused by mutations that decrease transforming growth factor-ß (TGF-ß) signaling. Although aneurysms develop throughout the arterial tree, the aortic root is a site of heightened risk. To identify molecular determinants of this vulnerability, we investigated the heterogeneity of vascular smooth muscle cells (VSMCs) in the aorta of Tgfbr1 M318R/+ LDS mice by single cell and spatial transcriptomics. Reduced expression of components of the extracellular matrix-receptor apparatus and upregulation of stress and inflammatory pathways were observed in all LDS VSMCs. However, regardless of genotype, a subset of Gata4-expressing VSMCs predominantly located in the aortic root intrinsically displayed a less differentiated, proinflammatory profile. A similar population was also identified among aortic VSMCs in a human scRNAseq dataset. Postnatal VSMC-specific Gata4 deletion reduced aortic root dilation in LDS mice, suggesting that this factor sensitizes the aortic root to the effects of impaired TGF-ß signaling.

Novel insights into molecular and immune subtypes of biliary tract cancers.

Biliary tract cancers (BTCs), which include cholangiocarcinoma (CCA) and gallbladder cancer (GBC), are heterogenous malignancies characterized by distinct molecular features often associated with specific clinical traits and/or outcomes. Such complex molecular heterogeneity, both within each BTC subtype and between distinct subtypes, poses a great challenge to personalized medicine. Recent technological advances have allowed the integration of multiple -omics derived from large cohorts of patients with distinct solid cancers to ultimately design stratification algorithms for prognostic prediction or more efficient treatment allocation. In this regard, although BTCs lag behind other tumors when it comes to our understanding of their molecular complexity, over the past decade, tremendous efforts have been made to generate supervised or unsupervised molecular classifications. As a result, CCAs and GBCs can be assigned to distinct molecular and/or prognostic classes. Notably, the discovery of biologically relevant subgroups of tumors harboring frequent targetable alterations (i.e., mutations in IDH1, FGFR2 fusion proteins) holds important therapeutic implications for BTCs, particularly iCCA. Furthermore, the recent application of single cell-based technologies or more conservative (and less precise) "virtual microdissection" algorithms to isolate signals derived from the immune and stromal cells has identified the first microenvironment-based classes. In this chapter, we will review the molecular and immune classes of BTCs, with a particular focus on their clinical implications.

The Molecular Pathogenesis and Targeted Therapies for Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a group of malignancies of the bile ducts with high mortality rates and limited treatment options. In the past decades, remarkable efforts have been dedicated toward elucidating the specific molecular signaling pathways and oncogenic loops driving cholangiocarcinogenesis to ultimately develop more effective therapies. Despite some recent advances, an extensive intra- and inter-tumor heterogeneity together with a poorly understood immunosuppressive microenvironment significantly compromises the efficacy of available treatments. Here, we provide a concise review of the latest advances and current knowledge of the molecular pathogenesis of CCA focusing on clinically relevant aberrations as well as future research avenues.

Postnatal Smad3 Inactivation in Murine Smooth Muscle Cells Elicits a Temporally and Regionally Distinct Transcriptional Response.

Heterozygous, loss of function mutations in positive regulators of the Transforming Growth Factor-ß (TGF-ß) pathway cause hereditary forms of thoracic aortic aneurysm. It is unclear whether and how the initial signaling deficiency triggers secondary signaling upregulation in the remaining functional branches of the pathway, and if this contributes to maladaptive vascular remodeling. To examine this process in a mouse model in which time-controlled, partial interference with postnatal TGF-ß signaling in vascular smooth muscle cells (VSMCs) could be assessed, we used a VSMC-specific tamoxifen-inducible system, and a conditional allele, to inactivate Smad3 at 6 weeks of age, after completion of perinatal aortic development. This intervention induced dilation and histological abnormalities in the aortic root, with minor involvement of the ascending aorta. To analyze early and late events associated with disease progression, we performed a comparative single cell transcriptomic analysis at 10- and 18-weeks post-deletion, when aortic dilation is undetectable and moderate, respectively. At the early time-point, Smad3-inactivation resulted in a broad reduction in the expression of extracellular matrix components and critical components of focal adhesions, including integrins and anchoring proteins, which was reflected histologically by loss of connections between VSMCs and elastic lamellae. At the later time point, however, expression of several transcripts belonging to the same functional categories was normalized or even upregulated; this occurred in association with upregulation of transcripts coding for TGF-ß ligands, and persistent downregulation of negative regulators of the pathway. To interrogate how VSMC heterogeneity may influence this transition, we examined transcriptional changes in each of the four VSMC subclusters identified, regardless of genotype, as partly reflecting the proximal-to-distal anatomic location based on in situ RNA hybridization. The response to Smad3-deficiency varied depending on subset, and VSMC subsets over-represented in the aortic root, the site most vulnerable to dilation, most prominently upregulated TGF-ß ligands and pro-pathogenic factors such as thrombospondin-1, angiotensin converting enzyme, and pro-inflammatory mediators. These data suggest that Smad3 is required for maintenance of focal adhesions, and that loss of contacts with the extracellular matrix has consequences specific to each VSMC subset, possibly contributing to the regional susceptibility to dilation in the aorta.

Distinct Contribution of Global and Regional Angiotensin II Type 1a Receptor Inactivation to Amelioration of Aortopathy in Tgfbr1 M318R/+ Mice.

Angiotensin II (Ang II) type 1 receptor (AT1R) signaling controls both physiological and pathogenetic responses in the vasculature. In mouse models of Loeys-Dietz syndrome (LDS), a hereditary disorder characterized by aggressive aortic aneurysms, treatment with angiotensin receptor blockers (ARBs) prevents aortic root dilation and associated histological alterations. In this study we use germline and conditional genetic inactivation of Agtr1a (coding for the AT1a receptor) to assess the effect of systemic and localized AT1R signaling attenuation on aortic disease in a mouse model of LDS (Tgfbr1 M318R/+). Aortic diameters and histological features were examined in control and Tgfbr1 M318R/+ mice with either germline or Mef2C SHF -Cre mediated genetic inactivation of Agtr1a, the latter resulting in deletion in second heart field (SHF)-derived lineages in the aortic root and proximal aorta. Both systemic and regional AT1R signaling attenuation resulted in reduction of diameters and improvement of tissue morphology in the aortic root of LDS mice; these outcomes were associated with reduced levels of Smad2/3 and ERK phosphorylation, signaling events previously linked to aortic disease in LDS. However, regional AT1a inactivation in SHF-derived lineages resulted in a more modest reduction in aortic diameters relative to the more complete effect of germline Agtr1a deletion, which was also associated with lower blood pressure. Our findings suggest that the therapeutic effects of AT1R antagonisms in preclinical models of aortic disease depend on both regional and systemic factors and suggest that combinatorial approaches targeting both processes may prove beneficial for aneurysm mitigation.

Insights on the Pathogenesis of Aneurysm through the Study of Hereditary Aortopathies.

Thoracic aortic aneurysms (TAA) are permanent and localized dilations of the aorta that predispose patients to a life-threatening risk of aortic dissection or rupture. The identification of pathogenic variants that cause hereditary forms of TAA has delineated fundamental molecular processes required to maintain aortic homeostasis. Vascular smooth muscle cells (VSMCs) elaborate and remodel the extracellular matrix (ECM) in response to mechanical and biochemical cues from their environment. Causal variants for hereditary forms of aneurysm compromise the function of gene products involved in the transmission or interpretation of these signals, initiating processes that eventually lead to degeneration and mechanical failure of the vessel. These include mutations that interfere with transduction of stimuli from the matrix to the actin-myosin cytoskeleton through integrins, and those that impair signaling pathways activated by transforming growth factor-ß (TGF-ß). In this review, we summarize the features of the healthy aortic wall, the major pathways involved in the modulation of VSMC phenotypes, and the basic molecular functions impaired by TAA-associated mutations. We also discuss how the heterogeneity and balance of adaptive and maladaptive responses to the initial genetic insult might contribute to disease.

Evaluating the therapeutic potential of ADAR1 inhibition for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other types of breast cancer that can be effectively treated by targeted therapies, no such targeted therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to prevent sensing of endogenous double-stranded RNAs. ADAR1 is highly expressed in breast cancer including TNBC. Here, we demonstrate that expression of ADAR1, specifically its p150 isoform, is required for the survival of TNBC cell lines. In TNBC cells, knockdown of ADAR1 attenuates proliferation and tumorigenesis. Moreover, ADAR1 knockdown leads to robust translational repression. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene expression. IFNAR1 reduction significantly rescued the proliferative defects of ADAR1 loss. These findings establish ADAR1 as a novel therapeutic target for TNBC tumors.