Masey Ross

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is characterized by prolonged severe constriction of the basilar artery, which often leads to ischemic brain damage. Locally elevated concentrations of spasmogenic substances induce persistent depolarization of myocytes in the basilar artery, leading to continuous influx of calcium (Ca) through voltage-sensitive Ca channels and myocyte contraction. Potassium (K) channel openers may have therapeutic utility to oppose membrane depolarization, dilate the arteries, and reduce ischemia. Here, we examined the involvement of vascular Kv7 K channels in the pathogenesis of cerebral vasospasm and tested whether Kv7 channel openers are effective therapeutic agents in a rat model of SAH. Patch-clamp experiments revealed that 3 different spasmogens (serotonin, endothelin, and vasopressin) suppressed Kv7 currents and depolarized freshly isolated rat basilar artery myocytes. These effects were significantly reduced in the presence of a Kv7 channel opener, retigabine. Retigabine (10 μM) also significantly blocked L-type Ca channels, reducing peak inward currents by >50%. In the presence of a selective Kv7 channel blocker, XE991, the spasmogens did not produce additive constriction responses measured using pressure myography. Kv7 channel openers (retigabine or celecoxib) significantly attenuated basilar artery spasm in rats with experimentally induced SAH. In conclusion, we identify Kv7 channels as common targets of vasoconstrictor spasmogens and as candidates for therapeutic intervention for cerebral vasospasm.

BACKGROUND: Physical activity (PA) is recommended for women with breast cancer (BC); however, data are sparse on the association of PA with quality of life (QOL) and patient-reported symptoms for women on adjuvant endocrine therapy (AET). METHODS: Women with hormone receptor-positive BC who were taking AET completed standardized surveys about their health-related QOL, AET-related symptoms, and levels of PA using validated measures. A Wald chi-square test and an analysis of variance were used to assess associations with PA and independent variables. Generalized linear regression analyses assessed associations between PA, QOL, and AET-related symptoms. RESULTS: The analytic cohort included 485 Black and White women. Black race, a high body mass index (BMI), and being on aromatase inhibitors (vs tamoxifen) were associated with lower PA in a bivariate analysis. In a multivariate analysis, lower self-reported PA was associated with a high BMI (P = .02) and chemotherapy uptake (P = .006). Better health-related QOL (P = .01), less severe overall AET-related symptoms (P = .02), and less severe gynecological symptoms (P = .03) were associated with increasing levels of moderate PA. CONCLUSIONS: Among women taking AET, moderate levels of PA may be associated with fewer medication-related symptoms and overall better ratings of health-related QOL. Because of the low levels of PA observed in the sample overall and particularly for Black women, identifying successful strategies to promote PA are needed.

BACKGROUND: Higher levels of tumor-infiltrating lymphocytes (TILs) in breast cancers are associated with increased likelihood of pathologic complete response (pCR) to chemotherapy. DNA methyltransferase inhibitors (DNMTi) can augment immune responses to cancers, decreasing myeloid-derived suppressor cells (MDSCs) and increasing T lymphocyte responsiveness. We have shown that the DNMTi decitabine augments the effectiveness of immunotherapy using murine triple-negative breast cancer (TNBC) models. The primary objective was to determine whether DNMTi+immune checkpoint blockade would increase stromal TIL (sTIL) in primary breast cancers before neoadjuvant chemotherapy (NCT). METHODS: ×4 doses over 5 days) followed by 2 doses of pembrolizumab (200 mg, 2 weeks apart)-before starting NCT. Biopsies before and after window immunotherapy quantified TILs and programmed death-ligand 1 (PD-L1) expression. Patients proceeded to NCT and tumor resection per standard of care. Mid-study, results of the KEYNOTE 522 trial led to patients with TNBC receiving additional pembrolizumab concurrently with standard NCT and in the adjuvant setting. RESULTS: 46 patients (median age 54.5 years, range 28-72; 71.7% white, 28.3% black; 100% female) were treated. 21 patients had TNBC and received neither neoadjuvant pembrolizumab concurrently with NCT nor adjuvant pembrolizumab (Cohort A), 7 patients had TNBC and did receive concurrent and/or adjuvant pembrolizumab (Cohort A2), and 18 patients were estrogen receptor positive and/or progesterone receptor positive and received neither concurrent nor adjuvant pembrolizumab (Cohort B). Blood samples collected after decitabine administration before pembrolizumab showed a 59% decrease (p<0.01) in monocytic MDSCs compared with baseline. 38 patients had paired biopsies for sTIL and 37 for PD-L1 evaluation. Cohorts A/A2 experienced an sTIL increase of 6.1% (p<0.008); Cohort B experienced an sTIL increase of 8.3% (p=0.006). PD-L1 expression increased by 73.9% (p<0.01). 14 of 43 patients (32.6%) who proceeded to resection achieved pCR (n=11 of 27 (40.1%) in Cohorts A/A2 and n=3 of 16 (18.8%) in Cohort B). The most frequently reported immune-related adverse events were adrenal insufficiency (AI) (n=6, 13.0%), maculopapular rash (n=3, 6.5%), and hypothyroidism (n=3, 6.5%). Five of the six AI instances were at least partially attributable to hypophysitis/pituitary dysfunction, and one remains uncertain. CONCLUSIONS: Treatment in the pre-neoadjuvant window with decitabine and pembrolizumab could sensitize breast cancers to standard NCT by recruitment of TILs to the tumor tissue. The treatment was well-tolerated. TRIAL REGISTRATION NUMBER: NCT02957968.

Abstract Acute myelogenous leukemia (AML) is characterized by frequent aberrations of the PI3K/AKT/mTOR axis steming from diverse mechanisms, including FLT3, Ras, and c-KIT mutations, among multiple others. In a previous report, we demonstrated that dual inhibition of the PI3K/mTOR axis and the BH3-mimetic ABT-737, which inhibits both Bcl-2 and Bcl-xL, but not Mcl-1, exhibits potent anti-leukemia activity both in vitro and in vivo (Cancer Res. 2013;73(4):1340-51). It has also been shown that while TORC1 inhibitors such as rapamycin can elicit rebound activation of AKT in various tumor cells, dual inhibition of TORC1/2 potently inhibits AKT activation at both Thr308 and Ser473 sites. In the present studies, we examined whether concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL would lead to enhanced anti-leukemia activity in AML cells. Notably, dual tetracycline-inducible Bcl-2/Bcl-xL knockdown markedly sensitized acute myeloid leukemia (AML) cells to the dual TORC1/2 inhibitor INK128 (ChemieTek) in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/Bcl-xL antagonist ABT-737 (AbbVie) sharply induced cell death in multiple AML cell lines, including TKI-resistant FLT3-ITD mutants as well as primary AML blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras among others, while exerting minimal toxicity toward normal hematopoietic CD34+ cells. Combined treatment was particularly active against CD34+/CD38-/CD123+ primitive leukemia progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal micro-environment (i.e., co-culture with HS-5 cells), suggesting that this strategy may circumvent the protective effects of bone marrow stromal cells, which are known to play an important role in leukemia cell survival as well as drug resistance. Notably, INK128 was very effective in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737-mediated apoptosis. Furthermore, ectopic expression of Mcl-1 dramatically attenuated INK128/ABT737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 interactions. In contrast to INK128, rapamycin increased AML cell AKT phosphorylation, and was largely ineffective in down-regulating Mcl-1, decreasing 4EBP1 phosphorylation, or enhancing ABT-737 lethality. Interestingly, addition of a specific AKT inhibitor to the rapamycin/ABT-737 regimen sharply increased apoptosis in association with pronounced AKT inactivation and Mcl-1 down-regulation, analogous to effects observed with INK128/ABT-737. These findings argue that AKT activation may oppose rapamycin/ABT-737 lethality in AML cells. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak shRNA knock-down reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic AML xenograft model (P &lt; 0.0001; log-rank test for combined treatment vs either agent alone). In addition, analysis of subcutaneous AML-derived tumors showed significant declines in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with in vitro results. Collectively, these findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in AML. They also raise the possibility that dual mTORC1/mTORC2 inhibitors may offer advantages over pure mTORC1 inhibitors in this setting. Disclosures No relevant conflicts of interest to declare.