Citations

Abstract Introduction: Small molecule therapeutics of estrogen receptor-positive/HER2-negative breast cancer remains an area of active investigation where novel agents are greatly needed for treatment of hormone therapy resistant metastatic disease. The biguanide hexyl-benzyl-biguanide (HBB) is a potent inhibitor of CYP3A4 arachidonic acid (AA) epoxygenase activity and inhibits breast cancer cell proliferation and MCF-7 breast cancer tumor growth in nude mice. To explore the impact of bioisosteric substitution of the benzyl moiety of HBB with a cubane moiety, we synthesized hexyl-(cuban-1-yl-methyl)-biguanide (HCB) and tested its potency for the inhibition of the cognate CYP3A4 target AA epoxygenase activity as well as breast cancer cell proliferation of hormone therapy sensitive and resistant cell lines. Results: HCB selectively inhibited CYP3A4-mediated biosynthesis of (±)-14,15-EET with an IC50 of 4.7±0.2 uM vs. 64.8±6.5 uM for 8,9-EET and 26.5±1.9 uM for 11,12-EET. At 24 hours, HCB inhibited proliferation of MCF-7 (ER+HER2-), BT474 (ER+HER2+) and MDA-MB-231 (ER-HER2-) cells at IC50 of 8.4±1.2, 11±1.3 and 15±0.9 uM, respectively. At 48 hours, HCB inhibited proliferation of aromatase inhibitor and fulvestrant resistant (LR,FR), and cyclin dependent kinase inhibitor (CDKi) palbociclib resistant (LR,FR,PR) MCF-7 cell lines; LR,FR MCF-7AC1 (IC50 =1.34±0.1 uM) and LR,FR,PR MCF-7AC1 (IC50 =1.64±0.2 uM). Addition of 14,15-EET (1 uM) partially rescues MCF-7 cells from HCB-mediated inhibition of proliferation. OXPHOS is promoted, in part, by EETs. HCB is a potent OXPHOS inhibitor and rapidly inhibits O2 consumption of the MCF-7 and ZR75 (ER+HER2-) cells in a dose-dependent fashion (P<0.05). HCB treatment (10 uM) reduces mitochondrial membrane potential to 57.4±15.3% (P<0.001) of vehicle control in MCF-7 cells. Treatment with HCB at 20 uM for 0.5 hour also causes mitochondrial swelling in MCF-7 cells. HCB (10 uM) activates AMPK within 0.5 hour and increases the level of phosphorylation from 2.4±0.3 to 25.1±6.0 folds in a time dependent fashion in MCF-7 cells from 0.5-24 hours. Conclusion: These results show that HCB inhibits proliferation of ER+HER2- breast cancer cells, in part through inhibition of OXPHOS and suppression of the CYP product 14,15-EET. This inhibition is highly active in hormonal therapy and CDKi resistant ER+HER2- breast cancer cells. These results suggest that HCB is a novel and potent biguanide that has potential to be developed for inhibition of hormone therapy resistant and CDKi resistant breast cancer. Citation Format: Zhijun Guo, Jianxun Lei, Kwon Ho Hong, Beverly Norris, Craig M. Flory, Swaathi Jayaraman, Connor McDermott, Elizabeth Ambrose, Irina Sevrioukova, Tom Poulos, Ilia Denisov, Stephen Sliga, Robert J. Schumacher, Gunda I. Georg, John R. Hawse, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB078.

Abstract Cancer cells increase lipid metabolism by up-regulation lipogenic enzymes such as stearoyl-CoA desaturase 1 (SCD1). It is the rate-limiting enzyme in the de novo synthesis of fatty acids (FAs), catalyzing the conversion and biosynthesis of saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). We developed SSI-4, a novel highly specific SCD1 inhibitor. The anti-tumor activity ofSSI-4 was examined against different cholangiocarcinoma (CCA) models that included human and mouseCCA cell lines, CCA patient-derived xenografts (PDX) NOD SCID gamma (NSG) mouse models, and CCA PDX derived organoids. Using cell proliferation assay, we examined twelve CCA cell lines and 4 were very sensitive to SSI-4 (1-40 nM IC50). The remaining CCA cell lines showed moderate to no sensitivity to SSI-4. Studies at the protein level have shown that SCD1 is present in both sensitive and non-sensitive cells. Thus, SCD1 must be present in sensitive cells but is not predictive of response to an SCD1 inhibitor. In an effort to identify biomarkers predicting response to therapy, we performed assays, such as lipid, lactate, or energy phenotype which identified elevated lactate and unsaturated fatty acid levels as well as a glycolytic energy phenotype. In vivo, CCA PDX models have demonstrated that SSI-4strongly inhibited tumor growth. Immunohistochemical analysis of CCA tumors revealed elevated glycolytic genes in sensitive cells and thus, a potential predictive biomarker. We are currently pursuing a better understanding of the relationship between glycolysis and fatty acid synthesis/metabolism. We are also testing novel combination therapy of SSI-4 with the standard of care and other drugs to develop novel treatments for CCA. Using CCA PDX derived organoids showed synergistic antitumor activity of SSI-4in combination with cancer standard of care drugs such as gemcitabine and cisplatin. Collectively, we expect to develop a biomarker-driven selection of CCA patients sensitive to SCD1 inhibitors and novel SSI-4 combination therapy leading to improved patient survival. Citation Format: Justyna J. Gleba, Laura A. Marlow, Erin E. Miller, James L. Miller, Aylin Alasonyalilar-Demirer, Yi Guo, Kabir Mody, Lewis R. Roberts, Steven R. Alberts, Mark J. Truty, Tushar C. Patel, John A. Copland. Defining stearoyl-CoA desaturase 1 as a molecular therapeutic target against cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1004.

Objective The slope ratio (SR) is a metric that uses maximal expiratory flow‐volume (MEFV) curves to quantify lung‐emptying dynamics. SR values between 0.5–2.5 indicate homogeneous lung‐emptying, while values >2.5 are suggestive of heterogeneous lung‐emptying. Albuterol and exercise are commonly used to improve airflow in patients with cystic fibrosis (CF); however, the effects of these treatments on lung‐emptying dynamics are unknown. We hypothesized that SR values would decrease following albuterol and/or exercise, and that these decreases would be more pronounced following albuterol‐with‐exercise. Methods Nine patients (25±2 yrs, n=4 male, 5 female) diagnosed (genotype or sweat chloride) with CF underwent three interventions: i) albuterol (2.5 mg in 3 mL saline), ii) 30 mins of exercise (50% peak wattage), and iii) albuterol and exercise together. MEFV curves (spirometry) were obtained at 10 time points: immediately before (Pre) and 15 minutes after (T15) interventions. Forced expiratory volume in 1 second (FEV 1 ) and forced expiratory flow for 25–75% (FEF 25–75 ) of the forced vital capacity (FVC) were calculated for each curve. SR was calculated along the MEFV curve as the ratio between the tangent and chord slopes at serial volumes (200 mL increments), and SR values from 80%‐20% FVC were averaged to produce a single SR for each MEFV curve. Results Compared to exercise, albuterol and albuterol‐with‐exercise elicited greater increases in FEV 1 (Pre: 3.00±0.26 vs 2.99±0.25 vs 3.06±0.23 L, p>0.05; T15: 3.00±0.27 vs 3.27±0.30 vs 3.34±0.28 L, p=0.03; exercise vs albuterol vs exercise‐with‐albuterol, for each comparison respectively), FEF 25–75 (Pre: 2.52±0.40 vs 2.53±0.40 vs 2.57±0.36 L/s, p>0.05; T15: 2.48±0.39 vs 3.04±0.54 vs 3.10±0.43 L/s, p=0.04), and FVC (Pre: 4.19±0.38 vs 4.17±0.38 vs 4.22±0.37 L, p>0.05; T15: 4.16±0.39 vs 4.35±0.38 vs 4.40±0.41 L, p=0.02), as well as greater decreases in SR (Pre: 1.51±0.15 vs 1.56±0.12 vs 1.58±0.12, p>0.05; T15: 1.54±0.15 vs 1.46±0.17 vs 1.43±0.17, p=0.02). Changes in FEV 1 , FEF 25–75 , FVC, and SR were not different between albuterol and albuterol‐with‐exercise conditions (p>0.05). Reductions in SR were related to increases in FEV 1 following interventions (r=−0.55, p<0.01). Conclusions In contrast to exercise, albuterol caused CF patients to exhibit greater increases in FEV 1 , FEF 25–75 , and FVC, along with greater decreases in SR. There was no additive effect of exercise on albuterol. Changes in FEV 1 and SR were inversely correlated. These data suggest that albuterol promotes bronchodilation and homogeneous lung‐emptying in CF patients, and that the SR metric is effective for assessing patterns of bronchodilation following administration of different bronchodilators. Support or Funding Information Supported by HL131151 (SEB) and Mayo Clinic Internal Funding This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

This study examined the plasma FFA, intramuscular long chain acylcarnitine (LCAC) fatty acids and imTG stores during basal and hyperinsulinemic conditions. Methods: Endurance trained (TR, n=8) and sedentary individuals (SED, n=15) were studied under overnight postabsorptive and euglycemic hyperinsulinemic clamp (1.0mU·kg FFM −1 ·min −1 ) conditions. FFA kinetics were measured using a 5‐h infusion of [U‐ 13 C]oleate, for labeling of imTG and LCAC pools. Muscle biopsies were performed to measure imTG and imLCAC enrichment at 1400 h after 5‐h of infusion. imTG concentrations were greater in TR than SED individuals (P<0.01). Table SED Trained Saline** Clamp Saline** Clamp plasma oleate FFA (mpe) 0.210±0.018* 0.271±0.036* 0.189±0.026* 0.361±0.050* imLCAC (mpe) 0.025±0.003 0.005±0.002 0.016±0.005 0.002±0.002 imTG (mpe) 0.018±0.003† 0.011±0.001† 0.024±0.004† 0.013±0.001† P<0.001 saline vs. clamp; *P<0.000 between compartment (plasma vs. imLCAC vs. imTG); †P<0.000 imLCAC and imTG Methods: Endurance trained (TR, n=8) and sedentary individuals (SED, n=15) were studied under overnight postabsorptive and euglycemic hyperinsulinemic clamp (1.0mU·kg FFM −1 ·min −1 ) conditions. FFA kinetics were measured using a 5‐h infusion of [U‐ 13 C]oleate, for labeling of imTG and LCAC pools. Muscle biopsies were performed to measure imTG and imLCAC enrichment at 1400 h after 5‐h of infusion. Conclusion: The imTG contributes more to the imLCAC pools than plasma FFA both in the basal and hyperinsulinemic state, and hyperinsulinemia suppressed oleate FFA entry into the imLCAC pools, more so in trained than untrained adults. Muscle fatty acid oxidation is largely via obligatory cycling of fatty acids through imTG or other intracellular complex lipid pools.

Objective Blood pressure (BP) reactivity to stress is predictive of the development of cardiovascular disease. Cardiovascular reactivity is attenuated in individuals after bariatric surgery; however, the underlying mechanisms are not completely understood. We hypothesized the BP response at the onset of isometric handgrip exercise would occur earlier and to a lesser degree in individuals who underwent bariatric surgery when compared to obese adults and the reliance on total peripheral resistance (TPR) would be attenuated. Methods Twenty‐six individuals [7 non‐obese (28±3 yrs, 23±1 kg/m 2 ), 11 obese (33±2 yrs, 39±1 kg/m 2 ), 8 post‐bariatric surgery (35±2 yrs, 29±1 kg/m 2 )] completed isometric handgrip exercise (40% maximum voluntary contraction) to exhaustion. Heart rate (HR, ECG) and arterial BP (brachial catheter) were measured continuously. Stroke volume was estimated from the pressure waveform and cardiac output (CO) and TPR were calculated. Peak change, time to peak, and rate of rise in BP were assessed during the first 30‐s of exercise. Results Compared to non‐obese controls, obese adults exhibited a slower initial rise in BP (9.3± 3.9 vs 21.7±2.5 s) and higher peak BP change (Δ6.7±2.1 vs Δ11.9±2.1 mmHg) at the onset of isometric handgrip exercise (p<0.05). Peak BP and the rate of rise were not different between individuals who underwent bariatric surgery (14.0±4.1 s, Δ5.6±1.3 mmHg) and non‐obese controls (p>0.05). Non‐obese controls exhibited an exercise‐mediated increase in CO whereas obese adults increased TPR (p<0.05). The increases in CO and TPR were less apparent in individuals who underwent bariatric surgery (p>0.05). Low cardiac baroreflex sensitivity (BRS) and heart rate variability (HRV: SDNN, standard deviation of normal to normal R‐R intervals) at rest were related to smaller increases in CO (BRS: R=0.47, p<0.01; SDNN: R=0.57, p<0.01) and greater increases in TPR (BRS: R=−0.49, p=0.01; SDNN: R=−0.60, p<0.01) during exercise. Conclusions In contrast to obese adults, individuals who underwent bariatric surgery exhibit a rapid rise in BP at the onset of isometric handgrip exercise. This rapid increase in BP is associated with a fall in TPR and results in lower peak BP at the onset of isometric exercise. These data suggest bariatric surgery attenuates BP reactivity to physical stress via improvements in the time‐course of hemodynamic responses and reflex autonomic adjustments. Support or Funding Information NIH HL130339, NIH HL083947, NIH DK82424, NIH UL1 RR024150, Mayo Clinic Department of Anesthesiology, Mayo Foundation for Medical Education and Research This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The Genitourinary Pathology Society (GUPS) undertook a critical review of the recent advances in bladder cancer focusing on important topics of high interest for the practicing surgical pathologist and urologist. This review represents the second of 2 manuscripts ensuing from this effort. Herein, we address the effective reporting of bladder cancer, focusing particularly on newly published data since the last 2016 World Health Organization (WHO) classification. In addition, this review focuses on the importance of reporting bladder cancer with divergent differentiation and variant (subtypes of urothelial carcinoma) histologies and the potential impact on patient care. We provide new recommendations for reporting pT1 staging in diagnostic pathology. Furthermore, we explore molecular evolution and classification, emphasizing aspects that impact the understanding of important concepts relevant to reporting and management of patients.

The Genitourinary Pathology Society (GUPS) undertook a critical review of the recent advances in bladder neoplasia with a focus on issues relevant to the practicing surgical pathologist for the understanding and effective reporting of bladder cancer, emphasizing particularly on the newly accumulated evidence post-2016 World Health Organization (WHO) classification. The work is presented in 2 manuscripts. Here, in the first, we revisit the nomenclature and classification system used for grading flat and papillary urothelial lesions centering on clinical relevance, and on dilemmas related to application in routine reporting. As patients of noninvasive bladder cancer frequently undergo cystoscopy and biopsy in their typically prolonged clinical course and for surveillance of disease, we discuss morphologies presented in these scenarios which may not have readily applicable diagnostic terms in the WHO classification. The topic of inverted patterns in urothelial neoplasia, particularly when prominent or exclusive, and beyond inverted papilloma has not been addressed formally in the WHO classification. Herein we provide a through review and suggest guidelines for when and how to report such lesions. In promulgating these GUPS recommendations, we aim to provide clarity on the clinical application of these not so uncommon diagnostically challenging situations encountered in routine practice, while also importantly advocating consistent terminology which would inform future work.

Abstract Immune checkpoint blockade (ICB) has revolutionized cancer therapy. However, the response of patients to ICB is difficult to predict. Here, we examined 81 patients with lung cancer under ICB treatment and found that patients with MET amplification were resistant to ICB and had a poor progression-free survival. Tumors with MET amplifications had significantly decreased STING levels and antitumor T-cell infiltration. Furthermore, we performed deep single-cell RNA sequencing on more than 20,000 single immune cells and identified an immunosuppressive signature with increased subsets of XIST- and CD96-positive exhausted natural killer (NK) cells and decreased CD8+ T-cell and NK-cell populations in patients with MET amplification. Mechanistically, we found that oncogenic MET signaling induces phosphorylation of UPF1 and downregulates tumor cell STING expression via modulation of the 3′-UTR length of STING by UPF1. Decreased efficiency of ICB by MET amplification can be overcome by inhibiting MET. Significance: We suggest that the combination of MET inhibitor together with ICB will overcome ICB resistance induced by MET amplification. Our report reveals much-needed information that will benefit the treatment of patients with primary MET amplification or EGFR–tyrosine kinase inhibitor resistant-related MET amplification. This article is highlighted in the In This Issue feature, p. 2659

PURPOSEThe purpose of this phase II study was to evaluate hyperthermic intraperitoneal chemotherapy (HIPEC) with carboplatin for recurrent ovarian cancer during secondary cytoreductive surgery.MATERIALS AND METHODSPatients were intraoperatively randomly assigned to carboplatin HIPEC (800 mg/m2for 90 minutes) or no HIPEC, followed by five or six cycles of postoperative IV carboplatin-based chemotherapy, respectively. Based on a binomial single-stage pick-the-winner design, an arm was considered winner if ≥ 17 of 49 patients were without disease progression at 24 months post-surgery. Secondary objectives included postoperative toxicity and HIPEC pharmacokinetics.RESULTSOf 98 patients, 49 (50%) received HIPEC. Complete gross resection was achieved in 82% of the HIPEC patients and 94% of the standard-arm patients. Bowel resection was performed in 37% of patients in the HIPEC arm compared with 65% in the standard ( P = .008). There was no perioperative mortality and no difference in use of ostomies, length of stay, or postoperative toxicity. At 24 months, eight patients (16.3%; 1-sided 90% CI, 9.7 to 100) were without progression or death in the HIPEC arm and 12 (24.5%; 1-sided 90% CI, 16.5 to 100) in the standard arm. With a medium follow-up of 39.5 months, 82 patients progressed and 37 died. The median progression-free survival in the HIPEC and standard arms were 12.3 and 15.7 months, respectively (hazard ratio, 1.54; 95% CI, 1 to 2.37; P = .05). There was no significant difference in median overall survival (52.5 v 59.7 months, respectively; hazard ratio, 1.39; 95% CI, 0.73 to 2.67; P = .31). These analyses were exploratory.CONCLUSIONHIPEC with carboplatin was well tolerated but did not result in superior clinical outcomes. This study does not support the use of HIPEC with carboplatin during secondary cytoreductive surgery for platinum-sensitive recurrent ovarian cancer.