Citations

Abstract Rationale: MUC5B and MUC5AC are neighboring genes on chromosome 11p15 that encode the two predominant mucins secreted in the respiratory airways. Increased airway mucin concentrations and an imbalance between these two mucins drive COPD symptoms and the development of airflow obstruction or COPD, but the epigenomic regulation of this loci has not been characterized in the airways. Methods: We isolated DNA from bronchial airway brushings of 72 participants with >20 pack-years smoking history from the Subpopulations and Intermediate Outcomes Measures in COPD Study (SPIROMICS). 36 participants had GOLD-based moderate-to-severe COPD (FEV1/FVC<0.70, FEV1<80%) and 36 were without or with mild COPD. We used the Illumina Infinium Methylation EPIC BeadChip Array to assess proportion of DNA methylation (β) spanning 689,937 CpG sites. We evaluated proportional DNA methylation (β) at 96 CpG loci within a 103kB region using matrixQTL for cis-expression quantitative trait methylation (eQTM: 10kB flanks relative to MUC5AC/MUC5B) integrating bulk RNA-seq data and mQTL models integrating gene sequencing data to identify DNA variation associated with proportion eQTM methylation. Models included age, sex, BMI, clinical site, current smoking vs past smoking, total pack-years, three ancestral components of ancestry, chipset, and position. Results: We identified a top eQTM, cg00524486, marginally associated with MUC5B expression (coefficient estimate=8.75, punadjusted=0.0014, pFDR=0.065, Figure 1B). This eQTM was associated with CT scan parametric response mapping-based functional small airways disease (PRM-fSAD, beta coefficient[β]=3.64, p=0.011). We tested for DNA variation associated with methylation of this top eQTM which identified two methylation quantitative trait loci (mQTL: rs885454 [β=0.044, minor allele frequency[MAF]=0.09, pFDR=0.01], rs868902 [β=0.020, MAF=0.43, pFDR=0.08, Figure 1A) associated with cg00524486 methylation, of which the more frequent mQTL, rs868902, was marginally associated with PRM-fSAD (p=0.07) in the larger SPIROMICS cohort with MUC5B DNA sequencing data. Conclusions: This preliminary multi-omic study of the lower airway epithelium demonstrates that the expression of MUC5B is epigenetically regulated by neighboring CpG probe methylation (eQTM) represented by DNA variation (mQTL). These studies require further confirmation in a larger dataset of airway samples and support a role for epigenetic variation as a biologic contributor and biomarker of early COPD pathogenesis.

AbstractThis article investigates the performance of a commercial BeO optically stimulated luminescent (OSL) dosimetry system (myOSLchip, RadPro GmbH International, Remscheid, Germany) through the application of the commissioning framework for luminescent dosimeters as described in the American Association of Physicists in Medicine Task Group 191 (AAPM TG191) report. Initial clinical experiences and dosimetric results are also presented. The following properties of the system were characterized: linearity correction factors ranged from ‐0.5% to +3% for dose levels spanning 0.1 to 20 Gy. Beam quality correction factors (relative to 6 MV) ranged from ‐4.5% (2.5FFF) to +4.5% (15MV) for photon beams and +1.9% (6 MeV) to +4.3% (20 MeV) for electron beams. An average (µ) signal loss per reading of ‐2.13% ± 0.20% was measured, however greater signal loss was observed in the first reading (µ = ‐2.6% ± 0.46%). An initial decline in individual element sensitivity relative to baseline was observed from 0–15 Gy cumulative dose (µ = ‐1.98% ± 0.55%), with negligible further deterioration from 15–32 Gy (µ = ‐2.38% ± 0.85%). Post‐irradiation, there was a transient OSL signal which faded with a half‐life of 1.8 min; this signal enhancement was +5% at 5 min post‐irradiation and +1% at 15 min relative to 24 h. Dosimeter response was not dependent on average dose rate in the range of 100–2500 MU/min. With respect to clinical testing, equal or superior performance compared with aluminum oxide OSLs (nanoDots) is shown for a range of clinical techniques and modalities including TSET, TBI, en‐face electrons, and pacemaker/out‐of‐field measurements. The feasibility of myOSLchip to serve as a primary clinical in vivo dosimetry system and direct replacement for Landauer's microStar system is demonstrated.

Using 32 immune cell surface markers, we identified 23 monocyte clusters in murine blood. Diabetic gastroparesis was associated with a significant decrease in two circulating classical monocyte-like clusters and an upregulation of the Ccr2-Ccl2 axis in the gastric muscularis propria, suggesting increased tissue monocyte migration. This study offers new targets by pointing to a possible role for two classical monocyte subsets connected to the Ccr2- Ccl2 axis.

Splenic marginal zone lymphoma (SMZL) is a B-cell malignancy with a typically indolent course. However, some patients have more aggressive disease and initial treatment can range from observation to chemoimmunotherapy. The reason for this spectrum of disease behavior is incompletely understood but the tumor microenvironment (TME) likely plays a role as recent studies have shown that an optimized antitumor immune response in SMZL patients would allow the immune system to better target malignant B cells and irradicate the disease. Regulatory CD4+T cells (Tregs) are crucial in maintaining TME homeostasis by suppressing effector T cells and malignant B cells and in this study, we therefore explored the key factors influencing the function and development of intratumoral Tregs in SMZL patients. We identified two distinct Treg subsets in the TME of SMZL patients: CD26+Tregs and CD161+Tregs, each with unique phenotypic profiles, functions, and prognostic significance. CD161+Tregs suppress CD8+T cells and malignant B cells more effectively than CD26+Tregs, which only inhibit CD8+T cells. Clinically, patients with inferior outcomes have significantly higher numbers of CD26+Tregs and fewer CD161+Tregs. To delineate differences between these subsets, we performed Cellular Indexing of Transcriptomes and Epitopes Sequencing (CITE-seq) on biopsies from 7 SMZL patients. We found that cytokine-inducible SH2-containing protein (CISH) is the most highly expressed gene in CD26+Tregs compared to CD161+Tregs, aside from FOXP3. CISH has been reported to be a negative regulator of immune effector cell activation in tumor models. Although CISH deficiency leads to a loss of Foxp3 expression and reduced Treg suppressive function, the mechanism by which CISH modulates Treg function, especially immune suppression in hematologic malignancies, remains largely unknown. To further investigate the mechanism of CISH in Treg function in MZL patients, we conducted CITE-seq analysis on a larger cohort of MZL patients (n=12), divided into groups based on outcomes (EFS24 failed, n=4; EFS24 achieved, n=8). The CITE-seq analysis results showed significantly higher CISH expression in Tregs from patients with inferior outcomes (P=1.07E-06). We then explored factors influencing CISH expression and found that TCR engagement and treatment with cytokines (IL-15, IL-2, and IL-7) elevated CISH expression in Tregs. While we previously observed that lymphoma B cells could enhance Foxp3 expression in CD4+CD25-T cells, this study we found that B cells could also enhance CISH and CD26 expression in CD4+CD25-T cells in vitro, indicating that a high frequency of B cells in the environment may lead to increased CISH expression in Treg cells leading to an unfavorable outcome in patients. We further investigated the role of CISH in the function of Treg cells. Following CISH knockdown in Treg cells, we observed decreased CD26 expression and reduced suppressive function on CD8+ T cell proliferation. Using CRISPR-Cas9, we knocked out CISH in the Karpas 299 cell line, a human T cell lymphoma line that constitutively expresses CD25, Foxp3, and CISH. CISH-KO Karpas 299 cells showed significantly less apoptosis and Foxp3 expression compared to CISH-WT cells. Bulk RNA-seq analysis of CISH-KO and CISH-WT Karpas 299 cells revealed that CISH-KO cells showed enhanced DNA replication and cell cycle pathway activity. Taken together, our results suggest that high expression of CISH in Tregs may predict an unfavorable outcome in SMZL patients. Malignant B-cells in the TME of SMZL patients may promote expression of CD26 and CISH in Tregs, thereby modulating Treg function. Targeting CISH could decrease the immunosuppressive activity of Tregs in the TME, potentially optimizing antitumor immunity in SMZL patients.

Abstract Waldenstrom macroglobulinemia (WM) is a lymphoplasmacytic lymphoma associated with an increase in serum immunoglobulin M (IgM) monoclonal protein. The underlying causes of the disease are still not fully understood, but cells in the bone marrow (BM) microenvironment (TME) are anticipated to play a significant role in disease progression. We previously reported that myeloid-derived suppressor cells (MDSCs), a phenotypically heterogeneous group of cells known for their ability to suppress immune responses, are expanded in the bone marrow of patients with WM and that the expansion of MDSCs is particularly notable for increased numbers of polymorphonuclear MDSCs (PMN-MDSCs). In the present study, we evaluated the ability of WM cells to attract MDSCs to the tumor microenvironment (TME). We also evaluated whether PMN-MDSCs suppress the cytotoxic function of T-cells and thereby promote the survival of WM cells. Our study confirms the expansion of MDSCs, notably PMN-MDSCs in BM of WM patients as compared to normals. We also found that PMN-MDSCs expressing CD66b was a predominant population in WM patients. To determine the phenotype and transcriptome of both PMN-MDSCs and monocytic MDSCs (M-MDSCS) in WM, we compared BM aspirates from WM patients to normal bone marrow (NBM) and performed cellular indexing of transcriptomes and epitopes by sequencing (CITEseq) analysis (WM=3, NBM=2) followed by qPCR validation. We found that MDSCs in WM express significantly higher levels of HIF1-α overall (p<0.02) compared to NBM. Furthermore, PMN-MDSCs are more abundant in WM bone marrow than M-MDSCs and express significantly higher levels of CXCR4 (p<0.001), myeloperoxidase (MPO; p<0.0003), and azuricidin (AZU1; p<0.0004), suggesting an activated phenotype. To determine whether WM cells attract MDSCs to the BM, we performed a transwell assay and showed that WM cells (BCWM.1 cell line) attracted MDSCs, particularly PMN-MDSCs, to a greater extent than M-MDSCs (n=3; p<0.001). We next evaluated whether MDSCs prevent cytotoxic T-cells from lysing WM cells. In-vitro co-culture studies with BCWM.1 cells and T-cytotoxic cells (B:CD8-T) showed that T-cytotoxic cells were able to lyse the WM cell line by 46.4% , (median range 46.4 - 47.8%, p=0.001). However, when BCWM.1 cells and T-cytotoxic cells were co-cultured in the presence of MDSCs (i.e B:CD8-T:M), the specific lysis of BCWM.1 cells was reduced by 12.6% respectively (range 2.2- 12.6%, p<0.001). Furthermore, we observed that B:CD8-T:M co-culture significantly reduced CD8-T cell activation as compared to activated T cells alone (17.9%) (median range 17.8- 18%, p<0.001). In contrast, no affect was observed with B: CD8-T coculture by 91% (range 90.6- 91.4% p<.001) confirming that MDSCs suppress CD8-T cell activation. Our study therefore provides evidence that MDSCs in WM have an activated phenotype and that PMN-MDSCs in WM are attracted by malignant cells in the bone marrow. Furthermore, we find that the increased presence of PMN-MDSCs in the WM bone marrow environment suppresses T-cell lysis of malignant cells and may thereby contribute to disease progression. These findings provide a strong rationale for targeting MDSCs as a potential therapeutic strategy in WM.

Microglia help limit the progression of Alzheimer’s disease (AD) by constraining amyloid-β (Aβ) pathology, effected through a balance of activating and inhibitory intracellular signals delivered by distinct cell surface receptors. Human leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory receptor of the immunoglobulin (Ig) superfamily that is expressed on myeloid cells and recognizes apolipoprotein E (ApoE) among other ligands. Here, we find that LILRB4 is highly expressed in the microglia of patients with AD. Using mice that accumulate Aβ and carry a transgene encompassing a portion of the LILR region that includes LILRB4 , we corroborated abundant LILRB4 expression in microglia wrapping around Aβ plaques. Systemic treatment of these mice with an anti-human LILRB4 monoclonal antibody (mAb) reduced Aβ load, mitigated some Aβ-related behavioral abnormalities, enhanced microglia activity, and attenuated expression of interferon-induced genes. In vitro binding experiments established that human LILRB4 binds both human and mouse ApoE and that anti-human LILRB4 mAb blocks such interaction. In silico modeling, biochemical, and mutagenesis analyses identified a loop between the two extracellular Ig domains of LILRB4 required for interaction with mouse ApoE and further indicated that anti-LILRB4 mAb may block LILRB4-mApoE by directly binding this loop. Thus, targeting LILRB4 may be a potential therapeutic avenue for AD.

Abstract The role of the bone marrow (BM) microenvironment in regulating the antitumor immune response in Waldenstrom macroglobulinemia (WM) remains limited. Here we transcriptionally and phenotypically profiled non-malignant (CD19− CD138−) BM cells from WM patients with a focus on myeloid derived suppressive cells (MDSCs) to provide a deeper understanding of their role in WM. We found that HLA-DRlowCD11b+CD33+ MDSCs were significantly increased in WM patients as compared to normal controls, with an expansion of predominantly polymorphonuclear (PMN)-MDSCs. Single-cell immunogenomic profiling of WM MDSCs identified an immune-suppressive gene signature with upregulated inflammatory pathways associated with interferon and TNF signaling. Gene signatures associated with an inflammatory and immune suppressive environment were predominately expressed in PMN-MDSCs. In vitro WM PMN-MDSCs demonstrated robust T-cell suppression, and their viability and expansion was notably enhanced by G-CSF and TNFα. Furthermore, BM malignant B-cells attracted PMN-MDSCs to a greater degree than monocytic MDSCs. Collectively, these data suggest that malignant WM B cells actively recruit PMN-MDSCs which promote an immunosuppressive BM microenvironment through a direct T cell inhibition, while release of G-CSF/TNFα in the microenvironment further promotes PMN-MDSC expansion and in turn immune suppression. Targeting PMN-MDSCs may therefore represent a potential therapeutic strategy in patients with WM.

Abstract The stiffening of the extracellular matrix (ECM), a phenomenon that often promotes metastasis and immunosuppression in many cancers, is a process that is not yet fully understood. Our study reveals that ATR, a molecule primarily recognized for its role in maintaining the genome, plays a crucial role in ECM stiffness-induced immune evasion and metastasis in triple negative breast cancer (TNBC) through DNA repair independent manner. In response to ECM stiffness, ATR activates SUN2, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. This activation enhances the interaction of SUN2 with outer nuclear membrane proteins KASH1 and KASH2. The interaction between SUN2 and KASH1/2 facilitates the nuclear localization of β-catenin, which in turn promotes epithelial mesenchymal transition (EMT) and reduces the expression of E-cadherin. E-cadherin is essential for attracting CD103+ immune cells that perform anti-tumor immunity. Interestingly, inhibiting ATR reduces EMT in tumor cells, leading to an increase in CD103+ dendritic cell infiltration while decreasing neutrophil and polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration. Further, we found that ATR levels are inversely correlated with immunotherapy response rates in patients. The alteration in immune cell composition sensitizes tumors to immunotherapy, thereby suppressing tumor growth and metastasis. In conclusion, the role of ATR in remodeling the immune environment could be crucial for optimizing the clinical use of ATR inhibitors. This makes ATR a promising target for overcoming immunotherapy resistance. We suggest early or prolonged treatment with an ATR inhibitor to potentially reduce metastasis rates and improve the prognosis of TNBC patients. Furthermore, we suggest using ATR levels as a marker to predict response rates and prognosis in TNBC patients. For patients with higher ATR expression levels, combination treatment with ATR inhibitors and immunotherapy may yield better outcomes. Enhanced anti-tumor immunity could increase the chances for patients to eliminate cancer cells and help end breast cancer. Finally, our research suggests the combined treatment of ATR inhibition and immunotherapy for patients without necessarily applying DNA damage stimulation. This approach could potentially reduce toxicity while still benefiting patients. Citation Format: Xinyi Tu, Zhenkun Lou, Robert Mutter. ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5366.