Characterizing the Genomic Landscape of the Micropapillary Subtype of Urothelial Carcinoma of the Bladder Harboring Activating Extracellular Mutations of ERBB2.

The micropapillary subtype of urothelial carcinoma (MPUC) of the bladder is a very aggressive histological variant of urothelial bladder cancer (UBC). A high frequency of MPUC contains activating mutations in the extracellular domain (ECD) of ERBB2. We sought to further characterize ERBB2 ECD-mutated MPUC to identify additional genomic alterations that have been associated with tumor progression and therapeutic response. In total, 5,485 cases of archived formalin-fixed, paraffin-embedded UBC underwent comprehensive genomic profiling to identify ERBB2 ECD-mutated MPUC and evaluate the frequencies of genomic co-alterations. We identified 219 cases of UBC with ERBB2 ECD mutations (74% S310F and 26% S310Y), of which 63 (28.8%) were MPUC. Genomic analysis revealed that TERT, TP53, and ARID1A were the most common co-altered genes in ERBB2-mutant MPUC (82.5%, 58.7%, and 39.7%, respectively) and did not differ from ERBB2-mutant non-MPUC (86.5%, 51.9%, and 35.3%). The main differences between ERBB2 ECD-mutated MPUC compared with non-MPUC were KMT2D, RB1, and MTAP alterations. KMT2D and RB1 are tumor-suppressor genes. KMT2D frequency was significantly decreased in ERBB2 ECD-mutated MPUC (6.3%) in contrast to non-MPUC (27.6%; P < .001). RB1 mutations were more frequent in ERBB2 ECD-mutated MPUC (33.3%) than in non-MPUC (17.3%; P = .012). Finally, MTAP loss, an emerging biomarker for new synthetic lethality-based anticancer drugs, was less frequent in ERBB2 ECD-mutated MPUC (11.1%) than in non-MPUC (26.9%; P = .018). Characterizing the genomic landscape of MPUC may not only improve our fundamental knowledge about this aggressive morphological variant of UBC but also has the potential to identify possible prognostic and predictive biomarkers that may drive tumor progression and dictate treatment response to therapeutic approaches.

Cancer cell plasticity and MHC-II-mediated immune tolerance promote breast cancer metastasis to lymph nodes.

Tumor-draining lymph nodes (TDLNs) are important for tumor antigen-specific T cell generation and effective anticancer immune responses. However, TDLNs are often the primary site of metastasis, causing immune suppression and worse outcomes. Through cross-species single-cell RNA-Seq analysis, we identified features defining cancer cell heterogeneity, plasticity, and immune evasion during breast cancer progression and lymph node metastasis (LNM). A subset of cancer cells in the lymph nodes exhibited elevated MHC class II (MHC-II) gene expression in both mice and humans. MHC-II+ cancer cells lacked costimulatory molecule expression, leading to regulatory T cell (Treg) expansion and fewer CD4+ effector T cells in TDLNs. Genetic knockout of MHC-II reduced LNM and Treg expansion, while overexpression of the MHC-II transactivator, Ciita, worsened LNM and caused excessive Treg expansion. These findings demonstrate that cancer cell MHC-II expression promotes metastasis and immune evasion in TDLNs.

Addition of Losartan to FOLFIRINOX and Chemoradiation Reduces Immunosuppression-Associated Genes, Tregs, and FOXP3+ Cancer Cells in Locally Advanced Pancreatic Cancer.

PURPOSE: Adding losartan (LOS) to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) resulted in 61% R0 surgical resection in our phase II trial in patients with locally advanced pancreatic cancer (LAPC). Here we identify potential mechanisms of benefit by assessing the effects of neoadjuvant LOS on the tumor microenvironment. EXPERIMENTAL DESIGN: We performed a gene expression and immunofluorescence (IF) analysis using archived surgical samples from patients treated with LOS+FFX+CRT (NCT01821729), FFX+CRT (NCT01591733), or surgery upfront, without any neoadjuvant therapy. We also conducted a longitudinal analysis of multiple biomarkers in the plasma of treated patients. RESULTS: In comparison with FFX+CRT, LOS+FFX+CRT downregulated immunosuppression and pro-invasion genes. Overall survival (OS) was associated with dendritic cell (DC) and antigen presentation genes for patients treated with FFX+CRT, and with immunosuppression and invasion genes or DC- and blood vessel-related genes for those treated with LOS+FFX+CRT. Furthermore, LOS induced specific changes in circulating levels of IL-8, sTie2, and TGF-ß. IF revealed significantly less residual disease in lesions treated with LOS+FFX+CRT. Finally, patients with a complete/near complete pathologic response in the LOS+FFX+CRT-treated group had reduced CD4+FOXP3+ regulatory T cells (Tregs), fewer immunosuppressive FOXP3+ cancer cells (C-FOXP3), and increased CD8+ T cells in pancreatic ductal adenocarcinoma lesions. CONCLUSIONS: Adding LOS to FFX+CRT reduced pro-invasion and immunosuppression-related genes, which were associated with improved OS in patients with LAPC. Lesions from responders in the LOS+FFX+CRT-treated group had reduced Tregs, decreased C-FOXP3 and increased CD8+ T cells. These findings suggest that LOS may potentiate the benefit of FFX+CRT by reducing immunosuppression.

FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in brain. We determine that this phenotype is an adaptation to decreased lipid availability in brain relative to other tissues, resulting in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Bevacizumab improves tumor infiltration of mature dendritic cells and effector T-cells in triple-negative breast cancer patients.

A single dose of bevacizumab reduced the density of angiopoietin-2-positive vessels while improving the infiltration of CD4+ T and CD8+ T cells, and mature dendritic cells in patients with primary triple-negative breast cancer. Our findings provide a rationale for including bevacizumab during neoadjuvant treatment to enhance the efficacy of immune checkpoint blockers in this disease.

Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma (GBM) trials. Here, we show that regulatory T (Treg) cells play a key role in GBM resistance to ICBs in experimental gliomas. Targeting glucocorticoid-induced TNFR-related receptor (GITR) in Treg cells using an agonistic antibody (αGITR) promotes CD4 Treg cell differentiation into CD4 effector T cells, alleviates Treg cell-mediated suppression of anti-tumor immune response, and induces potent anti-tumor effector cells in GBM. The reprogrammed GBM-infiltrating Treg cells express genes associated with a Th1 response signature, produce IFNγ, and acquire cytotoxic activity against GBM tumor cells while losing their suppressive function. αGITR and αPD1 antibodies increase survival benefit in three experimental GBM models, with a fraction of cohorts exhibiting complete tumor eradication and immune memory upon tumor re-challenge. Moreover, αGITR and αPD1 synergize with the standard of care treatment for newly-diagnosed GBM, enhancing the cure rates in these GBM models.

Author Correction: Non-invasive monitoring of chronic liver disease via near-infrared and shortwave-infrared imaging of endogenous lipofuscin.

A Correction to this paper has been published: https://doi.org/10.1038/s41551-020-0569-y.

Non-invasive monitoring of chronic liver disease via near-infrared and shortwave-infrared imaging of endogenous lipofuscin.

Monitoring the progression of non-alcoholic fatty liver disease is hindered by a lack of suitable non-invasive imaging methods. Here, we show that the endogenous pigment lipofuscin displays strong near-infrared and shortwave-infrared fluorescence when excited at 808 nm, enabling label-free imaging of liver injury in mice and the discrimination of pathological processes from normal liver processes with high specificity and sensitivity. We also show that the near-infrared and shortwave-infrared fluorescence of lipofuscin can be used to monitor the progression and regression of liver necroinflammation and fibrosis in mouse models of non-alcoholic fatty liver disease and advanced fibrosis, as well as to detect non-alcoholic steatohepatitis and cirrhosis in biopsied samples of human liver tissue.

Inducible ApoE gene repair in hypomorphic ApoE mice deficient in the low-density lipoprotein receptor promotes atheroma stabilization with a human-like lipoprotein profile.

OBJECTIVE: To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)-lipoprotein levels. APPROACH AND RESULTS: Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression (Apoe(h/h)Ldlr(-/-)Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non-high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6C(high) monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. CONCLUSIONS: Restoring apoE expression in Apoe(h/h)Ldlr(-/-)Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non-high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6C(high) monocytes levels and genetic reprogramming of lesional macrophages.

Apolipoprotein E4 domain interaction accelerates diet-induced atherosclerosis in hypomorphic Arg-61 apoe mice.

OBJECTIVE: Apolipoprotein (apo) E4 is an established risk factor for atherosclerosis, but the structural components underlying this association remain unclear. ApoE4 is characterized by 2 biophysical properties: domain interaction and molten globule state. Substituting Arg-61 for Thr-61 in mouse apoE introduces domain interaction without molten globule state, allowing us to delineate potential proatherogenic effects of domain interaction in vivo. METHODS AND RESULTS: We studied atherosclerosis susceptibility of hypomorphic Apoe mice expressing either Thr-61 or Arg-61 apoE (ApoeT(h/h) or ApoeR(h/h)mice). On a chow diet, both mouse models were normolipidemic with similar levels of plasma apoE and lipoproteins. However, on a high-cholesterol diet, ApoeR(h/h) mice displayed increased levels of total plasma cholesterol and very-low-density lipoprotein as well as larger atherosclerotic plaques in the aortic root, arch, and descending aorta compared with ApoeT(h/h) mice. In addition, evidence of cellular dysfunction was identified in peritoneal ApoeR(h/h) macrophages which released lower amounts of apoE in culture medium and displayed increased expression of major histocompatibility complex class II molecules. CONCLUSIONS: These data indicate that domain interaction mediates proatherogenic effects of apoE4 in part by modulating lipoprotein metabolism and macrophage biology. Pharmaceutical targeting of domain interaction could lead to new treatments for atherosclerosis in apoE4 individuals.

ApoE suppresses atherosclerosis by reducing lipid accumulation in circulating monocytes and the expression of inflammatory molecules on monocytes and vascular endothelium.

OBJECTIVE: We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. METHODS AND RESULTS: We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoe(h/h)Ldlr(-/-)) were compared to Apoe(-/-)Ldlr(-/-) mice. Despite 4-fold more plasma apoE than WT mice, Apoe(h/h)Ldlr(-/-) mice displayed similar plasma cholesterol as Apoe(-/-) Ldlr(-/-) mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoe(h/h)Ldlr(-/-) mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoe(h/h)Ldlr(-/-) mice had less circulating leukocytes and proinflammatory Ly6C(high) monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoe(h/h)Ldlr(-/-) mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. CONCLUSIONS: Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.