Thompson, Michael A

Traditionally associated with neuronal structure and function, neurotrophins (NTs; e.g. brain derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3 )) and their receptors (tropomyosin related kinase; Trk) have been found in the adult lung, pulmonary artery and airway; however, their roles remain unclear. Based on previous findings of pulmonary hypertension in NT receptor knockout mice, we hypothesized that NTs induce production of nitric oxide (NO) in pulmonary endothelial cells (EC), thereby modulating pulmonary vascular tone. In human pulmonary artery EC loaded with the NO sensitive fluorescent dye DAF‐2, 50 ƒÝM ATP and both BDNF and NT3 (1 and 10 nM) acutely (<5 min) and substantially in increased fluorescence levels. Addition of the NO scavenger PTIO reversed these changes. Specificity of DAF2 for NO was tested using the short‐acting NO donor MAHMA‐NONOate, and the nitrous oxide donor Sulfa‐NONOate. Furthermore, NT‐induced elevation in NO was significantly reduced by pre‐exposure to the tyrosine kinase inhibitor K252A (100 nM, 20 min), the NOS inhibitors L‐NMMA or L‐NAME, and the Ca2+ chelator BAPTA. Acute NT exposure also increased eNOS phosphorylation. These results indicate that NT¡¦s such as BDNF and NT3 can acutely modulate endothelial NO production, and may contribute to pulmonary vasodilation. Supported by NIH grants HL088029, (YSP), HL56470 (YSP), and Mayo Graduate School (LWM).

Regulation of intracellular Ca2+ in human airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca2+ release/reuptake and plasma membrane (PM) Ca2+ influx, including store‐operated Ca2+ entry (SOCE) following SR Ca2+ depletion. Studies in human ASM have shown that siRNA suppression of stromal interaction molecule 1 (STIM1) and Orai1 decrease SOCE; however, their exact role in regulating Ca2+ are still under investigation. In human ASM, STIM1 and STIM2 were found exclusively in SR membrane, and Orai1 only in PM (especially caveolae). GFP‐tagged STIM1 transfection showed intracellular/SR localization at baseline, with increased peri‐PM aggregation following store depletion with 10ƒÝM cyclopiazonic acid (CPA). Such aggregation was prevented by microtubule inhibition (nocodazole), but not actin inhibition (phalloidin). FRET analysis revealed co‐localization of STIM1 with TRPC3 and Orai1 in CPA‐stimulated cells. In fura‐2 loaded ASM cells, SOCE was significantly reduced by STIM1 and Orai1 siRNA (less so by STIM2 siRNA). However, basal Ca2+ and agonist (ACh, histamine)‐induced Ca2+ responses were substantially modulated by STIM2 suppression. These results indicate that in human ASM, STIM1 mediates SR‐PM interactions in regulating SOCE, while STIM2 modulates sub‐maximal agonist‐induced changes in Ca2+. Supported by NIH grant HL74309 (GCS), HL090595 (CMP) and HL088029 (YSP).

Plasma membrane Ca 2+ influx and sarcoplasmic reticulum (SR) Ca 2+ release are key components of intracellular Ca 2+ regulation in airway smooth muscle (ASM). We and others have previously found that ASM Ca 2+ regulation involves propagating Ca 2+ oscillations via these mechanisms. Ca 2+ oscillations in non‐excitable cells involve influx via arachidonic acid (AA) regulated channels (ARC); however, little is known about ARC in ASM. In human ASM cells loaded with fura2 (5 μM), we found that 10 μM AA triggered Ca 2+ oscillations. OAG, oleic acid and palmitic acid (10 μM) failed to elicit Ca 2+ responses. Pre‐incubation with LaCl 3 1 μM and 1mM; 5 min) inhibited AA‐induced oscillations. Inhibition of SR Ca 2+ release (ryanodine 10 μM and IP3 channel inhibitor Xestospongin C 10 μM; 30 min), decreased Ca 2+ oscillation frequency and amplitude. Inhibitors of receptor‐operated channels (SKF‐96365; 10 μM), lipoxygenase (zileuton; 10 μM), or cyclooxygenase (indomethacin; 1 μM), did not affect oscillations. Finally, siRNA for Caveolin‐1, STIM1 and Orai3 (25nM) reduced frequency and amplitude of AA‐induced oscillations. These results indicate a novel caveolar Ca 2+ influx mechanism in human ASM mediated via AA that interacts with SR to produce repetitive SR Ca 2+ release. Such AA‐mediated influx could potentially contribute to increased Ca 2+ in ASM during inflammation. Supported by NIH grants HL090595 (CMP) and HL088029 (YSP)

Abstract Background: Overall, patients with cancer experience a greater risk of adverse outcomes following SARS-CoV-2 infection; however, little is known for those with BC. Methods: CCC19 (NCT04354701) is an international cohort study aimed at investigating the impact of COVID-19 in patients with a history of or active cancer using de-identified data on patient demographics, cancer history, clinical course and outcomes of COVID-19. The current analysis includes patients from U.S. and Canada with invasive BC and laboratory-confirmed SARS-CoV-2 entered between March 17, 2020 and July 2, 2020. Co-primary outcomes were hospitalization during COVID-19 illness and 30-day all-cause mortality. Frequencies for categorical variables and medians (range) for continuous variables were estimated. Final presentation will include bivariable and multivariable Cox proportional hazards regression analysis to identify risk factors (including BC subtypes and therapies, for which data collection is ongoing) associated with 30-day all-cause mortality and severe COVID-19 illness (composite of any hospitalization requiring supplemental oxygen, admission to an intensive care unit [ICU], use of mechanical ventilation, or death). Results: During the study period, a total of 2683 patients with cancer and COVID-19 were accrued in the CCC19 registry including 529 (20%) with invasive BC. Among patients with BC, 352 (67%) were 60 years or older; 518 (98%) were women; 275 (52%) non-Hispanic White, 116 (22%) non-Hispanic Black, 70 (13%) Hispanic, 56 (11%) in other categorizations; 178 (34%) had a smoking history; 75 (14%) ECOG performance status ≥2; 191 (36%) with &gt;2 active comorbidities; 64 (12%) with AJCC stage IV disease at BC diagnosis; and 267 (50%) were on anti-cancer treatment including systemic therapy, radiation, or surgery within 3 months of COVID-19 diagnosis. At least 323 (61%) patients with BC had 30-day follow-up after COVID-19 diagnosis, and 35 (7%) had 90-day follow-up. COVID-19 illness at initial diagnosis required outpatient care in 288 (54%), inpatient care in 193 (36%), and ICU care in 40 (8%). Overall, 247 (47%) were hospitalized and 30-day all-cause mortality was 9%. 30-day all-cause mortality rates by receipt of major BC treatment modalities (within past 3 months) were: 10% for those on cytotoxic systemic therapy vs 5% and 12% for noncytotoxic systemic therapy and local therapy, respectively. The table shows hospitalization and mortality outcomes by major demographic and BC treatment strata. The final presentation will incorporate the latest patient accrual and evaluate independent clinical risk factors associated with serious COVID-19 outcomes in patients with BC. Conclusions: This represents the largest study to date of COVID-19 outcomes in patients with invasive BC. Nearly half of the patients with BC required hospitalization during their COVID-19 disease course and we observed a 9% 30-day all-cause mortality. Submitted on behalf of the COVID-19 and Cancer Consortium (ccc19us.org) Table 1. COVID-19 related hospitalization and 30-day all-cause mortality for all patients with invasive BCNAny Hospitalization30-day all-cause MortalityN% [95% CI]N% [95% CI]Total population52924747 [42-51]499 [7-12]Age&lt;601774425 [19-32]21 [0-4]60-691155144 [35-54]87 [3-13]70-791206655 [46-64]1714 [8-22]80+1178674 [65-81]2219 [12-27]ECOG PS0-132212238 [33-43]165 [3-8]2+756181 [71-89]1621 [13-32]Active Comorbidities0741014 [7-23]00 [0-5]1-22239442 [36-49]178 [5-12]&gt;219112968 [60-74]2915 [10-21]Treatment IntentCurative1866434 [28-42]95 [2-9]Palliative743953 [41-64]1216 [9-27]Cancer StatusRemission/NED33115447 [41-52]247 [5-11]Active disease, stable or responding to treatment1194840 [31-50]76 [2-12]Active disease, progressing372773 [56-86]1130 [16-47]Treatment Modality (within 3 months)Cytotoxic chemotherapy833542 [31-54]810 [4-18]Noncytotoxic therapy1856435 [28-42]105 [3-10]Local therapy (surgery or radiation)341750 [32-68]412 [3-27] Citation Format: Ali Raza Khaki, Dimpy P Shah, Maryam B Lustberg, Melissa K Accordino, Daniel G Stover, Gayathri Nagaraj, Donna R Rivera, Edith A Perez, Sara M Tolaney, Jeffrey Peppercorn, Petros Grivas, Jeremy L Warner, Corrie A Painter, Gilberto de Lima Lopes, Jr, Solange Peters, Michael A Thompson, Toni K Choueiri, Brian I Rini, Gary H Lyman, Nicole M Kuderer, Shaveta Vinayak. Characteristics and outcomes of SARS-CoV-2 infection in patients with invasive breast cancer (BC) from the COVID-19 and cancer consortium (CCC19) cohort study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-01.

Abstract Despite unprecedented successes with immune checkpoint blockade in advanced cancers, these agents provide durable clinical benefit in only a subset of patients. In addition, the clinical management of patients receiving immune checkpoint blockade remains quite challenging due to the unpredictable and kinetically heterogeneous responses which can manifest as late responses, pseudoprogression, or hyperprogression in subsets of patients. The dynamics and heterogeneity of programmed cell death protein ligand 1 (PD-L1, aka B7-H1) expression confound its use as a predictive biomarker in cancer immunotherapy, but blood-based biomarkers have the potential to predict responders and detect mechanisms of resistance to immunotherapy. We previously reported that Bim (BH3-only protein) is a downstream signaling molecule of the PD-1 pathway, and that Bim levels in circulating tumor-reactive T-cells reflected patient responses to anti-PD-1 therapy in melanoma. We also discovered the existence of soluble PD-L1 (sPD-L1) in the plasma of cancer patients and showed a correlation of sPD-L1 with cancer stages in renal cell carcinoma and with prognosis in metastatic melanoma. Here we evaluated the frequency of tumor-reactive T cells with Bim expression along with effector memory profile and sPD-L1 as biomarkers of response in a new cohort of patients with metastatic melanoma and lung cancer undergoing anti-PD-1 therapy. We recruited 100 cancer patients treated with anti-PD-1 therapy who had peripheral blood collected at baseline and at each subsequent radiographic tumor evaluation. Frequencies of Bim+ and effector memory CD8+ T cells were measured by flow cytometry in gated tumor-reactive CD11a high PD1+ CD8+ T cells. We also measured levels of sPD-L1 in plasma at baseline and serially during treatment with ELISA. Baseline T cell markers and sPD-L1 levels and their percent changes in patients who had a radiographic response were compared to those who had progressive disease (PD). We found that patients with objective response after 4 cycles of anti-PD-1 therapy had higher frequency of Bim+ CD8+ T cells at baseline compared to patients with PD (mean 43% vs. 30%, P=0.0484). The frequencies of Bim+ T cells decreased significantly after the first 3 months of treatment in responders compared with progressors (mean -16% vs. + 40% P=0.0111). The frequency of effector memory CD8+ T cells also dramatically increased in responders in 3-6 months after anti-PD-1 therapy (mean 89% vs. 9.56%, p=0.002). High baseline sPD-L1 was associated with progression on anti-PD-1 therapy (mean 2.8 ng/mL vs. 0.7 ng/mL, p=0.07) or development of immune-related toxicities and the levels increased by 12 weeks in patients progressing on therapy. Conclusion: Measurements of T cell biomarkers (Bim and effector memory) and sPD-L1 levels provide a new noninvasive way to predict and monitor patient responses to anti-PD-1 monotherapy in melanoma and lung cancers. Note: This abstract was not presented at the conference. Citation Format: Roxana S. Dronca, Aaron Mansfield, Xin Liu, Susan Harrington, Elizabeth Enninga, Svetomir Markovic, Lisa Kottschade, Rob Mcwilliams, Matthew Block, Wendy Nevala, Michael Thompson, Haidong Dong. Blood-based T cell biomarkers and soluble PD-L1 predict responses and immune-related toxicity to PD-1 blockade in melanoma and lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A38.

Abstract Metastatic melanoma (MM) is the most aggressive type of skin cancer contributing to approximately 80% of all skin cancer related deaths. In 2016, it was estimated that more 76,000 cases would be diagnosed in the United States. The high metastatic potential and aggressive clinical behavior of this disease makes it a major health problem. As a consequence there has been notable development in for novel targeted (i.e. BRAF inhibitors) and immune therapies (i.e. anti-PD1 and CTLA4 inhibitors). However, despite improvements in patient outcomes, most patients develop resistance within 6-11 months on dual BRAF/MEK inhibition, and 5 months on immune therapy, highlighting the need to identify new therapies that improve disease management and patient survival. Increased expression of CyclinD1 has been reported to occur as a mechanism of resistance to BRAF inhibition, resulting in reactivation of the canonical activating pathway in melanoma, MAPK. Thus, we hypothesize that combined inhibition of CDK4/6 and MAPK signaling will provide a novel anti-tumor synergy drug combination superior to current standard of care in BRAF mutant patient-derived preclinical models. Using MM human tissue from patients we developed 3 BRAF mutant preclinical models investigating a novel triple therapy drug combination BRAFi/MEKi/CDK4/6i. Protein analysis and drug proliferation assays were used to design the novel combination. In vivo, we found statistically significant differences in tumor inhibition between the novel combination and the current standard of care. The triple therapy also showed promising results, in vivo, when assessed as a salvage therapy following tumor escape from BRAFi/MEKi combination treatment. Citation Format: Antoneicka L. Harris, Michael Thompson, Svetomir Markovic, William Durham, Louis Dawson, Daniel Small, John A. Copland. Tumor regression in novel triple therapy BRAF mutant metastatic melanoma patient-derived preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-130. doi:10.1158/1538-7445.AM2017-LB-130