Because abnormalities of mucosal immunity have been suggested in human IgA nephropathy, we examined the involvement of mucosal immunity in IgA deposition to the kidney in hyper IgA (HIGA) mice, which was established as a mouse model for human IgA nephropathy with hyperserum IgA. The number of surface IgA+B220− lymphocytes in the intestinal lamina propria (LP) of HIGA mice increased 2.7-fold at 30 wk of age as compared with those at 10 wk of age, whereas normal mice did not show such increase. The surface IgA+B220− LP lymphocytes spontaneously secreted IgA in culture. Morphological studies showed that the surface IgA+B220− lymphocytes of murine intestinal LP are identical with plasma cells (PCs). About 20% of IgA+B220− PC in LP expressed both Mac-1 and CD19, suggesting that they may derive from peritoneal B-1 cells. Cell cycle study on intestinal IgA-PCs using bromodeoxyuridine revealed no difference between HIGA mice and normal mice, suggesting that the high frequency of IgA-producing PCs in HIGA mice is not due to enhanced proliferation or prolonged survival of IgA-producing PCs in LP. In addition, IgA secretion into the gut lumen of HIGA mice decreased drastically (to one forth) with aging. These data suggest that the increased number of intestinal IgA-producing PCs and the down-regulation of IgA excretion into the intestinal lumen might synergistically contribute to the hyperserum IgA in HIGA mice and resultant IgA deposition to the kidney.

Introduction: The EGFR-A763_Y764insFQEA is a unique exon 20 insertion mutation (w5% to 6% of exon 20 insertions), which, at the structural and enzyme kinetic level, more closely resembles EGFR tyrosine kinase inhibitor (TKI)sensitizing mutants, such as EGFR exon 19 indels and L858R. A limited number of preclinical models and clinical reports have studied the response of this mutant to EGFR TKIs. Methods: We used models of EGFR-A763_Y764insFQEA and more typical EGFR exon 20 insertion mutations to probe representative first-(gefitinib, erlotinib), second-(afatinib), third-generation (osimertinib), and in-development EGFR exon 20-specific (poziotinib, mobocertinib [TAK-788]) TKIs. We also compiled outcomes of EGFR-A763_Y764insFQEAmutated lung cancers treated with EGFR TKIs. Results: Cells driven by EGFR-A763_Y764insFQEA were consistently sensitive to EGFR TKIs (as opposed to those driven by typical EGFR exon 20 insertions [A767_V769du-pASV, D770_N771insSVD and H773_V774insH]), which were only inhibited by in-development EGFR TKIs at doses below those affecting wild-type EGFR. Most case instances (62.5% [95% confidence interval: 39%-86%], n ¼ 16) with lung cancers harboring EGFR-A763_Y764insFQEA responded to clinically available EGFR TKIs (including osimertinib) and to in-development EGFR exon 20-specific TKIs (including mobocertinib) with prolonged periods of progression-free survival in some cases. Median overall survival for EGFR TKI-treated cases was 22 months (95% confidence interval: 16-25). Mechanisms of acquired TKI resistance of this mutant remain underreported, but do seem to align with those of common mutations. Conclusions: To our knowledge, this is the largest report to confirm that the EGFR-A763_Y764insFQEA mutation is sen-*Corresponding author. Disclosure: Dr. Costa reports receiving personal fees (consulting fees and honoraria) and nonfinancial support (institutional research support) from Takeda/Millennium Pharmaceuticals, AstraZeneca, and Pfizer; and nonfinancial support (institutional research support) from Merck Sharp and Dohme Corporation, Merrimack Pharmaceuticals, Bristol-Myers Squibb, Clovis Oncology, Spectrum Pharmaceuticals, and Tesaro, all outside of the submitted work. Dr. Rangachari reports receiving nonfinancial support (institutional research support) from Bristol-Myers Squibb, Novocure, and AbbVie/Stemcentrx, all outside of the submitted work. Dr. VanderLaan reports receiving personal fees (consulting fees and honoraria) from Gala Therapeutics, Flatiron Health, Caris Life Sciences, and Foundation Medicine, all outside of the submitted work. Dr. Kobayashi reports receiving research support from Boehringer Ingelheim, MiNA Therapeutics, and Taiho Therapeutics; and personal fees (honoraria) from Boehringer Ingelheim, Bristol-Myers Squibb, and Takeda Pharmaceuticals outside of the submitted work. The remaining authors declare no conflict of interest.

Tissue factor initiates the extrinsic coagulation pathway by activating coagulation factor X to factor Xa, and factor V is a cofactor for the prothrombin activation by factor Xa. As factor Xa is known to promote the proliferation of mesangial cells in culture, the roles of the coagulation pathway and factor Xa were studied in an animal model of mesangioproliferative glomerulonephritis (MsPGN). MsPGN was induced in Wistar rats by an intravenous injection of anti-Thy 1.1 monoclonal antibody, OX-7. To clarify the role of factor Xa in MsPGN, a specific factor Xa inhibitor, DX-9065a, was injected intravenously at 2.5 or 10 mg/kg at the same time as OX-7, and kidney involvement was assessed by immunohistological analyses. We also examined p44/42 mitogen-activated protein (MAP) kinase activation. Time-course study revealed that expressions of tissue factor, factor V, and protease-activated receptor 2 (PAR2) were peaked on day 3, followed by factor X accumulation and mesangial proliferation. DX-9065a treatment significantly ameliorated proteinuria in a dose-dependent manner on day 8. Histological analyses showed a significant reduction in the size of glomeruli, the total number of glomerular cells, and crescent formation by DX-9065a treatment. Macrophage infiltration, which was rapidly observed on day 1 in disease control rats was not inhibited on days 1-3 by DX-9065a treatment, however it was suppressed on days 5-8. The deposition of fibrin, the number of PCNA-positive cells, and phosphorylation of p44/42 MAP kinase were markedly increased in the disease control group, whereas they were significantly reduced in the treatment group. Tissue factor and factor V induction may accelerate MsPGN through the activation and accumulation of factor X via proinflammatory and procoagulant mechanisms, and the inhibition of factor Xa would be a promising method to regulate the disease process.

Tumor heterogeneity underlies resistance to tyrosine kinase inhibitors (TKI) in lung cancers harboring EGFR mutations. Previous evidence suggested that subsets of preexisting resistant cells are selected by EGFR-TKI treatment, or alternatively, that diverse acquired resistance mechanisms emerge from drug-tolerant persister (DTP) cells. Many studies have used bulk tumor specimens or subcloned resistant cell lines to identify resistance mechanism. However, intratumoral heterogeneity can result in divergent responses to therapies, requiring additional approaches to reveal the complete spectrum of resistance mechanisms. Using EGFR-TKI-resistant cell models and clinical specimens, we performed single-cell RNA-seq and single-cell ATAC-seq analyses to define the transcriptional and epigenetic landscape of parental cells, DTPs, and tumor cells in a fully resistant state. In addition to AURKA, VIM, and AXL, which are all known to induce EGFR-TKI resistance, CD74 was identified as a novel gene that plays a critical role in the drug-tolerant state. In vitro and in vivo experiments demonstrated that CD74 upregulation confers resistance to the EGFR-TKI osimertinib and blocks apoptosis, enabling tumor regrowth. Overall, this study provides new insight into the mechanisms underlying resistance to EGFR-TKIs. Significance: Single-cell analyses identify diverse mechanisms of resistance as well as the state of tolerant cells that give rise to resistance to EGFR tyrosine kinase inhibitors.