Additional Mechanisms Of Action Research Articles

Efficacy of the residential care transition module: A telehealth intervention for dementia family caregivers of relatives living in residential long-term care settings.

The purpose of this study was to evaluate the efficacy of the Residential Care Transition Module, a six-session, psychosocial, and psychoeducational telehealth intervention for family caregivers of cognitively impaired relatives living in a residential long-term care setting. Eligible participants (including care recipients, regardless of time since admission) were randomized to treatment or usual care control conditions. Survey data were collected at baseline, 4 months, 8 months, and 12 months (N = 240). Primary analytic outcomes included caregiver subjective stress (a stress process mechanism) and depressive symptoms (a measure of global well-being). Secondary analytic outcomes included secondary role strains, residential care stress, caregiver sense of competence, and self-efficacy (additional mechanisms of action). General linear models tested for the main effects of the intervention at 4 months, and longitudinal mixed models examined the 12-month effects of the intervention. Post hoc analyses also examined the influence of moderators. No significant differences between the treatment and control groups for any primary analytic outcome were apparent. Caregivers in the treatment group whose relatives were admitted to residential long-term care in the prior 3 months were more likely to indicate reductions in depressive symptoms over the first 4 months of participation. Over the 12-month study period, caregivers in the treatment group who were employed reported increased self-efficacy over time. The heterogeneity of dementia care requires a broader consideration of key contextual factors that may influence the efficacy of nonpharmacological interventions. Aligning measures with the preferences, goals, and values of dementia caregivers may further demonstrate the direct benefits of interventions such as the Residential Care Transition Module. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Phase separation-competent FBL promotes early pre-rRNA processing and translation in acute myeloid leukaemia.

RNA-binding proteins (RBPs) are pivotal in acute myeloid leukaemia (AML), a lethal disease. Although specific phase separation-competent RBPs are recognized in AML, the effect of their condensate formation on AML leukaemogenesis, and the therapeutic potential of inhibition of phase separation are underexplored. In our in vivo CRISPR RBP screen, fibrillarin (FBL) emerges as a crucial nucleolar protein that regulates AML cell survival, primarily through its phase separation domains rather than methyltransferase or acetylation domains. These phase separation domains, with specific features, coordinately drive nucleoli formation and early processing of pre-rRNA (including efflux, cleavage and methylation), eventually enhancing the translation of oncogenes such as MYC. Targeting the phase separation capability of FBL with CGX-635 leads to elimination of AML cells, suggesting an additional mechanism of action for CGX-635 that complements its established therapeutic effects. We highlight the potential of PS modulation of critical proteins as a possible therapeutic strategy for AML.

Abstract 7225: Combination JAK1/2 and CDK8/19 inhibition demonstrates enhanced efficacy in myeloproliferative neoplasms

Abstract Myeloproliferative neoplasms (MPNs) are characterized by aberrant activation of the JAK-STAT pathway. Ruxolitinib (RUX) is a JAK1/2 inhibitor used to treat myelofibrosis (MF) and hydroxyurea-resistant/intolerant polycythemia vera. While RUX has demonstrated important clinical benefits, a large proportion of patients have persistent disease manifestations despite therapy. CDK8 regulates phosphorylation of STAT proteins in a RUX-independent manner; thus, CDK8 inhibition may alter downstream STAT target gene expression. RVU120 is a highly selective and potent CDK8/19 inhibitor that inhibits STAT1/5 phosphorylation. We hypothesized that the combination of RUX and RVU120 would act in a cooperative manner to attenuate JAK-STAT signaling and reduce MPN phenotypes in vivo. Using JAK2V617F mutant leukemic cell lines, we identified dose-responsive changes in viability with RUX and RVU120 in all cell lines. Pharmacodynamic analysis validated that STAT5 protein phosphorylation was decreased by RUX/RVU120, although at different phosphorylation sites by RUX (pTYR694) and RVU120 (pSER726/731); exposure to RUX+RVU120 inhibited phosphorylation at both sites. Notably, analysis of combinatorial effects of RUX+RVU120 on viability demonstrated drug synergies (combinatorial index <1) in RUX-naïve and resistant SET-2 cells. Nascent RNA expression data exploring JAK-STAT and mediator-related pathways of pathogenesis will be presented at the meeting. For in vivo studies, we used two previously-described murine models of MF and PV. Briefly, retrovirally-transfected (MSCV-MPLW515L-IRES-GFP) or Jak2V617F-conditional knock-in bone marrow (BM) was intravenously injected into lethally-irradiated congenic recipients. Mice were randomized and treated for several weeks with vehicle (VEH), single-agent RUX/RVU120, or RUX+RVU120 arms. Spleen weights between VEH and RUX/RVU120 treated mice at time of terminal sacrifice were significantly reduced across both models (p⇐0.009). In MPLW515L mice, WBCs and PB GFP% were significantly reduced (p<0.002) and histopathology demonstrated near-elimination of BM reticulin fibrosis and improved trilineage hematopoiesis between RUX+RVU120 and RUX arms. Data on overall survival, cytokine profiles, and the impact of RVU120 on dynamic gene expression will be presented at the meeting. The combination of RVU120 and RUX demonstrated biochemical synergy and differential inhibition of STAT5 phosphorylation in vitro. Further, in vivo treatment with RVU120/RUX+RVU120 resulted in significant reductions of disease manifestation (splenomegaly, WBC, fibrosis scoring, hematopoiesis) when compared to VEH/RUX. These data nominate JAK1/2 and CDK8/19 inhibition as a potential novel therapeutic strategy in MPNs; further work on nascent RNA expression and cytokine profiles will potentially elucidate additional mechanisms of action. Citation Format: Zachary Zaroogian, Elżbieta Adamczyk, Adrianna Moszyńska, Marta Obacz, Urszula Pakulska, Benjamin Durham, Milena Mazan, Shoron Mowla, Katarzyna Wnęk, Amritha Varshini Hanasoge Somasundara, Beata Barczuk, Aniela Gołas, Ross Levine, Tomasz Rzymski, Raajit Rampal. Combination JAK1/2 and CDK8/19 inhibition demonstrates enhanced efficacy in myeloproliferative neoplasms [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 7225.

Electrophysiological effects of kappa-opioid analgesic, RU-1205, using machine learning methods

The study is focused to the investigation of a new kappa-opioid agonist RU-1205, which exhibits an analgesic effect without causing dysphoric or aversive actions. It is assumed that this effects may be due to its functional selectivity, or the presence of an additional mechanism of action that involves blocking p38 mitogen-activated protein kinase (MAPK).The aim of the study was an experimental identification of RU-1205 mechanisms of action associated with the inhibition of MAPK p38 and functional selectivity for kappa opioid receptors.Materials and methods. The LFP activity was recorded in the male rats weighing 260–280 g (n=62) and implanted with chronic cortical and deep electrodes, after the intracerebroventricular administration of the well-studied reference substances: the selective kappa-opioid agonist U-50488 100 μg; the MAPK p38 blocker SB203580 1 μg; and the investigational compound RU-1205 at 350 μg. The weighted phase lag index (WPLI) was calculated. Subsequently, machine learning methods were employed to reduce the dimensionality and extract connectivity features using the principal component analysis method, then a signal classification was performed (models based on Gaussian processes). Using the local patch-clamp technique in the “whole-cell” configuration, the spike activity of pyramidal neurons in the basolateral amygdala was studied. Neurons were identified by their accommodation properties. After local perfusion of the test compounds, 3 dose-response curves were obtained for: (1) U-50488 at concentrations ranging from 0.001 to 10 μM; (2) combinations of U-50488 (0.001–10 μM) and RU-1205 (10 μM); and (3) the combinations of U-50488 (0.01–10 μM) and RU-1205 (100 μM).Results. The developed models made it possible to classify the compound RU-1205 as a “non-inhibitor” of MAPK p38 with a high probability. The results obtained were confirmed in patch-clamp experiments on acute brain slices where it was demonstrated that U-50488 statistically significantly increases the spike activity of pyramidal neurons of the basolateral amygdala (p <0.05), and RU-1205 interacts with U-50488, competitively suppressing its effect on the spike activity of neurons.Conclusion. The findings suggest that compound RU-1205 displays properties consistent with a functional kappa agonist activity and does not have a significant effect on MAPK p38. The study demonstrates the possibility of integrating electrophysiological measurements and advanced data analysis methods for a deep understanding of drug action and underscores the potential for further research in this area.

OP22 Topical Sphingosine-1-Phosphate (S1P) Receptor 1 Modulation Regulates Gut Angiogenesis in Inflammatory Bowel Diseases

Abstract Background S1P receptor (S1PR)1/5 modulators have been tested successfully in clinical development programs in ulcerative colitis (UC), leading to their FDA approval. They are believed to act through lymphocyte redistribution in peripheral lymphoid tissue and reduced numbers of circulating lymphocytes, but evidence from other organs suggests potential additional mechanisms of action. Methods A post-hoc analysis of the phase 3 trial of the S1PR1/5 modulator Ozanimod in patients with moderately to severely active UC was performed assessing an association of absolute lymphocyte count (ALC) with efficacy. Expression levels of S1PRs were analyzed in human intestinal tissues and primary cells using qPCR and/or immunohistochemistry (IHC). Effects of S1P, S1PR1/5 modulators and S1P1 knockdown on migration, proliferation, cytokine production, downstream signaling (immunoblot), tube formation of human intestinal microvascular endothelial cells (HIMEC) was evaluated. S1P and S1PR1/5 modulators were examined in a murine matrix plug angiogenesis model and with topical and systemic S1PR1/5 modulator therapy in dextrane sodium sulfate (DSS) colitis in vivo. Results Baseline ALC and relative or absolute ALC reductions were neither correlated with nor predictive of clinical or endoscopic outcomes. In murine DSS colitis topical therapy with a S1PR1/5 modulator ameliorated colitis but did not alter peripheral lymphocyte numbers. S1PR1 gene and protein expression was increased in UC and Crohn’s disease (CD) patients and predominantly expressed on HIMEC. S1P promoted HIMEC migration, proliferation, tube formation, IL-8 secretion, indicative of pro-angiogenic responses in vitro. Modulation of S1PR1/5 and knockdown of S1PR1 markedly inhibited these pro-angiogenic functions in the presence of high concentrations of S1P, but enhanced angiogenesis when low S1P concentrations were present. S1PR5 had no effect on angiogenesis. In the murine plug assay, S1PR1 induced angiogenesis. In acute DSS colitis, treatment of S1PR modulator by enema reduced blood vessels, but left lymphatics unaffected. Conclusion We present a novel mechanism of action of S1PR1 modulators in IBD intestinal tissue, potentially explaining a disconnect between ALC and efficacy observed in a post-hoc analysis of the phase 3 Ozanimod UC trial. The data presented suggest that S1P induced angiogenesis is attenuated by S1PR1 modulation in HIMEC from IBD patients when S1P levels comparable to blood were present. In the presence of S1P at concentrations consistent with lymph fluid angiogenesis was increased. Together with reduced numbers of circulating lymphocytes, this may contribute to efficacy of S1PR1 modulators in IBD patients.

New Steroidal Selenides as Proapoptotic Factors.

Cytostatic and pro-apoptotic effects of selenium steroid derivatives against HeLa cells were determined. The highest cytostatic activity was shown by derivative 4 (GI50 25.0 µM, almost complete growth inhibition after three days of culture, and over 97% of apoptotic and dead cells at 200 µM). The results of our study (cell number measurements, apoptosis profile, relative expression of apoptosis-related APAF1, BID, and mevalonate pathway-involved HMGCR, SQLE, CYP51A1, and PDHB genes, and computational chemistry data) support the hypothesis that tested selenosteroids induce the extrinsic pathway of apoptosis by affecting the cell membrane as cholesterol antimetabolites. An additional mechanism of action is possible through a direct action of derivative 4 to inhibit PDHB expression in a way similar to steroid hormones.

Subthalamic nucleus deep brain stimulation does not alter growth factor expression in a rat model of stable dopaminergic deficiency

BackgroundDeep brain stimulation (DBS) of the subthalamic nucleus (STN) has been a highly effective treatment option for mid-to-late-stage Parkinson’s disease (PD) for decades. Besides direct effects on brain networks, neuroprotective effects of STN-DBS – potentially via alterations of growth factor expression levels – have been proposed as additional mechanisms of action. ObjectiveIn the context of clarifying DBS mechanisms, we analyzed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) levels in the basal ganglia, motor and parietal cortices, and dentate gyrus in an animal model of stable, severe dopaminergic deficiency. MethodsWe applied one week of continuous unilateral STN-DBS in a group of stable 6-hydroxydopamine (6-OHDA) hemiparkinsonian rats (6-OHDASTIM) in comparison to a 6-OHDA control group (6-OHDASHAM) as well as healthy controls (CTRLSTIM and CTRLSHAM). BDNF and GDNF levels were determined via ELISAs. ResultsThe 6-OHDA lesion did not result in a persistent alteration in either BDNF or GDNF levels in a model of severe dopaminergic deficiency after completion of the dopaminergic degeneration. STN-DBS modestly increased BDNF levels in the entopeduncular nucleus, but even impaired BDNF and GDNF expression in cortical areas. ConclusionsSTN-DBS does not increase growth factor expression when applied to a model of completed, severe dopaminergic deficiency in contrast to other studies in models of modest and ongoing dopaminergic degeneration. In healthy controls, STN-DBS does not influence BDNF or GDNF expression. We consider these findings relevant for clinical purposes since DBS in PD is usually applied late in the course of the disease.

Abstract 1522: SGN-B6A induces immunogenic cell death as an additional mechanism of action

Abstract SGN-B6A is a novel investigational antibody-drug conjugate (ADC) directed to integrin beta-6 and uses the clinically validated vedotin drug-linker platform that delivers the microtubule disrupting agent, monomethyl auristatin E (MMAE). SGN-B6A is designed to bind and internalize the integrin beta-6/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. We have previously demonstrated the antitumor activity of SGN-B6A in cell line-derived xenograft models originating from multiple carcinomas as well as patient-derived xenograft models of non-small cell lung cancer (NSCLC). Other clinically validated vedotin ADCs that deliver MMAE have been shown to induce immunogenic cell death (ICD) in preclinical models and have demonstrated promising clinical activity in combination with immunotherapy. Since the induction of ICD appeared to be a consequence of the activity of MMAE, and is independent of the antibody that delivers it, we hypothesized that this mechanism of action may also apply to SGN-B6A. Consistent with this hypothesis, we observed that tumor cells treated with SGN-B6A in vitro showed key hallmarks of immunogenic cell death, including markers of endoplasmic reticulum (ER) stress, exposure of calreticulin, and release of ATP and high mobility group protein B1 (HMGB1). Further, in vivo studies demonstrated that treatment with SGN-B6A led to immune activation and recruitment of immune cells to the tumor environment. In an integrin beta-6-expressing syngeneic model, a vedotin ADC directed to integrin beta-6 has shown combinatorial activity with immunotherapy. Preclinical models suggest that, like other vedotin ADCs, SGN-B6A induces immunogenic cell death which then promotes activation and recruitment of immune cells to the tumor. We have recently reported promising single-agent activity of SGN-B6A in non-small cell lung, head and neck squamous cell, and esophageal cancer observed in interim results of a phase I study (NCT04389632). The combination of SGN-B6A with immunotherapy may be utilized as a potential treatment for integrin-beta-6-expressing tumors including NSCLC, head and neck squamous cell carcinoma, and esophageal carcinoma. Altogether, our preclinical and initial clinical results support the ongoing evaluation of SGN-B6A as a single agent and in combination with immune checkpoint inhibitors. Citation Format: Vivian H. Trang, Rebecca C. Mazahreh, John J. Gosink, Michelle Ulrich, Devra Olson, Allana Ubben, Sean Allred, Li-Ya Huang, Kelly Hensley, Piper M. Treuting, Kerry Klussman, Shaylin Higgins, Patrick Younan, Roma Yumul, Natalya Nazarenko, Robert P. Lyon. SGN-B6A induces immunogenic cell death as an additional mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1522.

Abstract 4423: XMT-2056, a HER2-targeted STING agonist antibody-drug conjugate, exhibits ADCC function that synergizes with STING pathway activation and contributes to anti-tumor responses

Abstract XMT-2056 is a systemically administered Immunosynthen STING agonist antibody-drug conjugate (ADC) that targets a novel HER2 epitope and induces complete tumor regressions with a single dose in multiple tumor models. We have previously shown that XMT-2056 delivers its STING agonist payload into tumor cells and FcγRI (CD64)-expressing myeloid cells, activating STING signaling in both cell types, leading to type I interferon (IFN) and anti-tumor innate immune responses. Here, we demonstrate that XMT-2056 exhibits ADCC (antibody-dependent cell-mediated cytotoxicity) function, which synergizes with STING pathway activation and induces potent cancer cell-killing activity in co-cultures of HER2-expressing cancer cells and FcγRIII+ (CD16+) immune cells. We show that both XMT-2056 and HT-19 (the unconjugated parental antibody) retain significant cancer cell-killing activity in an Fc-dependent manner in PBMC co-cultures depleted of FcγRI-expressing myeloid cells. This activity is abrogated by co-depletion of FcγRIII+ immune cells, illustrating the ADCC function of XMT-2056. In this setting, XMT-2056 cancer cell-killing activity was significantly increased compared to HT-19, suggesting that the STING payload contributes to the differential activity observed with XMT-2056 treatment. Indeed, co-treatment of cancer cell and immune cell co-cultures with HT-19 and free STING agonist payload enhanced the anti-tumor responses, although to a lesser extent than XMT-2056, suggesting a synergy between the ADCC function and STING pathway activation. Consistently, XMT-2056 treatment of HER2-expressing cancer cells co-cultured with unstimulated CD56+/FcγRIII+ NK cells induced granzyme b and IFN-γ cytokine production, expression of NK cell activation markers, and cancer cell-killing activity. The ADCC activity of HT-19 was comparable to that of trastuzumab in NK cell co-cultures. Finally, we found that depletion of FcγRI+ cells inhibited the cancer cell-killing activity of XMT-2056 in cancer cell and PBMC co-cultures more substantially compared to depletion of FcγRIII+ cells or CD56+ NK cells, indicating a greater contribution of myeloid cells to the XMT-2056 mechanism of action in this setting. Notably, XMT-2056 was capable of engaging both FcγRI+ myeloid cells and FcγRIII+ NK cells, activating both STING-mediated innate immune responses and ADCC function in triple cultures with HER2-expressing cancer cells. Collectively, our data reveal a synergy between ADCC function and STING pathway induction both mediated by XMT-2056, which enhances the cancer cell-killing activity of FcγRIII+ cells. This additional mechanism of action of XMT-2056 can potentially impact the overall anti-tumor immune responses in tumors infiltrated by FcγRIII+ cells. Citation Format: Jahna Soomer-James, Marc Damelin, Naniye Malli. XMT-2056, a HER2-targeted STING agonist antibody-drug conjugate, exhibits ADCC function that synergizes with STING pathway activation and contributes to anti-tumor responses. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4423.

Abstract 3103: Dual targeting MYC G-quadruplex and topoisomerase I by indenoisoquinolines for cancer therapy

Abstract MYC is one of the most important oncogenes and is overexpressed in a number of human cancers. The G-quadruplex formed in the MYC oncogene promoter (MycG4) is a transcriptional silencer and amenable to small molecule targeting. G-quadruplexes are noncanonical four-stranded DNA or RNA secondary structures that have emerged as novel therapeutic targets. Indenoisoquinolines are human topoisomerase I inhibitors with improved physicochemical and biological properties as compared to the clinically used camptothecin anticancer drugs. Three indenoisoquinolines, indotecan (LMP400), indimitecan (LMP776), and LMP744, have entered phase I clinical trials in adults with relapsed solid tumors and lymphomas. However, some indenoisoquinolines showed potent anticancer activity without being strong topoisomerase I inhibitors, suggesting additional mechanisms of action. Herein, we demonstrate that a large number of anticancer indenoisoquinolines strongly bind and stabilize MycG4 in vitro, using fluorescence, nuclear magnetic resonance (NMR), and circular dichroism (CD) spectroscopy, as well as gel electromobility shift assay (EMSA). In addition, MycG4-interactive indenoisoquinolines lower MYC mRNA and protein levels in vivo, as shown by western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments. Structure-activity-relationships of MycG4 recognition by indenoisoquinolines were investigated to understand structural requirements for binding. Systemic analysis of indenoisoquinoline derivatives led to the discovery of a synergistic effect of MYC inhibition and topoisomerase I inhibition on anticancer activity. Therefore, these results indicate that targeting the MYC promoter G-quadruplex to downregulate MYC is a new mechanism of action for anticancer indenoisoquinolines. Moreover, our data reveals dual targeting of MycG4 and topoisomerase I as a novel strategy for cancer therapy. Citation Format: Danzhou Yang, Mark Cushman, Kai-Bo Wang, Mohamed S. Elsayed, Guanhui Wu, Adam J. Buric, Mercedes DeMoss. Dual targeting MYC G-quadruplex and topoisomerase I by indenoisoquinolines for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3103.

Abstract 3253: CD30 is a marker of activated effector regulatory T cells in solid tumors providing clinical rationale for the combination of brentuximab vedotin and PD-1 inhibitors

Abstract Regulatory T cells (Tregs) play an important role in maintaining immune homeostasis by preventing excessive inflammation in normal tissues. In cancer, Tregs hamper antitumor immunosurveillance and may be a resistance mechanism to anti-PD-1 therapies. In preclinical cancer models, Treg depletion enhances antitumor cytotoxic immune responses, but systemic and persistent Treg removal can elicit immunopathology. Therapeutic strategies that selectively deplete highly suppressive activated intratumoral Tregs may avoid immune-related toxicities and amplify the activity of antitumor CD8 T cells during PD-1 blockade. CD30 is a member of the TNF receptor superfamily and is expressed by a subset of activated lymphocytes and various lymphomas. Here, transcriptomics and flow cytometry data from tumor and peripherally derived Tregs demonstrated CD30 is enriched on intratumoral Tregs. Among peripheral Tregs, CD30 expression was associated with an activated effector phenotype (FoxP3hiCD45RA-, fraction II). Further, treatment of dissociated NSCLC tumor spheroids with anti-PD-1 resulted in upregulated CD30 expression by Tregs but not intratumoral CD4 or CD8 T cells, which is consistent with Treg reactivation and supports CD30 as an activated Treg target. In vitro, CD30 expression on Tregs was dependent on signaling through the TCR and was significantly amplified by IL-2 and IL-15, suggesting CD30 may identify a subpopulation of activated antigen-specific Tregs. Brentuximab vedotin (BV) is an antibody-drug conjugate approved for clinical use in multiple types of lymphomas including cHL and PTCL. BV consists of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE). The activity of BV in lymphomas is thought to primarily result from tumor-directed intracellular MMAE release leading to apoptosis, though multiple immunomodulatory effects of BV have also been suggested. We have demonstrated through in vitro and in vivo preclinical model systems of human T cell activation that BV can selectively deplete CD30-expressing Tregs, amplifying cytotoxic CD8 T cell expansion. These and Treg CD30 expression data motivated the clinical exploration of BV plus pembrolizumab in metastatic PD-1 refractory melanoma and NSCLC (NCT04609566). Consistent with preclinical data, preliminary biomarker analysis from these studies showed a small but significant decrease in Tregs (CD4+CD25+CD127low/-) in peripheral blood after treatment with BV plus pembrolizumab. Further, limited on-treatment biopsies showed a trend toward increased intratumoral CD8:Treg (FoxP3) ratio in some patients receiving combination therapy. These data support the ongoing evaluation of BV in combination with PD-1 inhibitors in solid tumors and a potential additional mechanism of action for BV. Citation Format: Ryan A. Heiser, Bryan M. Grogan, Reice D. James, Michelle L. Ulrich, Jason D. Berndt, Brian P. O'Connor, Shyra J. Gardai, Hailing Lu, Scott M. Knowles. CD30 is a marker of activated effector regulatory T cells in solid tumors providing clinical rationale for the combination of brentuximab vedotin and PD-1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3253.

Use of a gene expression signature to identify trimetazidine for repurposing to treat bipolar depression.

The aim of this study was to repurpose a drug for the treatment of bipolar depression. A gene expression signature representing the overall transcriptomic effects of a cocktail of drugs widely prescribed to treat bipolar disorder was generated using human neuronal-like (NT2-N) cells. A compound library of 960 approved, off-patent drugs were then screened to identify those drugs that affect transcription most similar to the effects of the bipolar depression drug cocktail. For mechanistic studies, peripheral blood mononuclear cells were obtained from a healthy subject and reprogrammed into induced pluripotent stem cells, which were then differentiated into co-cultured neurons and astrocytes. Efficacy studies were conducted in two animal models of depressive-like behaviours (Flinders Sensitive Line rats and social isolation with chronic restraint stress rats). The screen identified trimetazidine as a potential drug for repurposing. Trimetazidine alters metabolic processes to increase ATP production, which is thought to be deficient in bipolar depression. We showed that trimetazidine increased mitochondrial respiration in cultured human neuronal-like cells. Transcriptomic analysis in induced pluripotent stem cell-derived neuron/astrocyte co-cultures suggested additional mechanisms of action via the focal adhesion and MAPK signalling pathways. In two different rodent models of depressive-like behaviours, trimetazidine exhibited antidepressant-like activity with reduced anhedonia and reduced immobility in the forced swim test. Collectively our data support the repurposing of trimetazidine for the treatment of bipolar depression.

The Endothelial Glycocalyx as a Target of Excess Soluble Fms-like Tyrosine Kinase-1.

Soluble fms-like tyrosine kinase-1 (sFlt-1) is a secreted protein that binds heparan sulfate expressed on the endothelial glycocalyx (eGC). In this paper we analyze how excess sFlt-1 causes conformational changes in the eGC, leading to monocyte adhesion, a key event triggering vascular dysfunction. In vitro exposure of primary human umbilical vein endothelial cells to excess sFlt-1 decreased eGC height and increased stiffness as determined by atomic force microscopy (AFM). Yet, structural loss of the eGC components was not observed, as indicated by Ulex europaeus agglutinin I and wheat germ agglutinin staining. Moreover, the conformation observed under excess sFlt-1, a collapsed eGC, is flat and stiff with unchanged coverage and sustained content. Functionally, this conformation increased the endothelial adhesiveness to THP-1 monocytes by about 35%. Heparin blocked all these effects, but the vascular endothelial growth factor did not. In vivo administration of sFlt-1 in mice also resulted in the collapse of the eGC in isolated aorta analyzed ex vivo by AFM. Our findings show that excess sFlt-1 causes the collapse of the eGC and favors leukocyte adhesion. This study provides an additional mechanism of action by which sFlt-1 may cause endothelial dysfunction and injury.

Emergence of Canonical and Noncanonical Genomic Variants following In Vitro Exposure of Clinical Mycobacterium tuberculosis Strains to Bedaquiline or Clofazimine.

In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected. We hypothesized that in vitro generation of bedaquiline- or clofazimine-resistant mutants could provide insight into additional mechanisms of action. We performed whole-genome sequencing and determined phenotypic MICs for both drugs on progenitor and mutant progenies. Mutants were induced through serial passage on increasing concentrations of bedaquiline or clofazimine. Rv0678 variants were identified in both clofazimine- and bedaquiline-resistant mutants, with concurrent atpE SNPs occurring in the latter. Of concern was the acquisition of variants in the F420 biosynthesis pathway in clofazimine-resistant mutants obtained from either a fully susceptible (fbiD: del555GCT) or rifampicin mono-resistant (fbiA: 283delTG and T862C) progenitor. The acquisition of these variants possibly implicates a shared pathway between clofazimine and nitroimidazoles. Pathways associated with drug tolerance and persistence, F420 biosynthesis, glycerol uptake and metabolism, efflux, and NADH homeostasis appear to be affected following exposure to these drugs. Shared genes affected by both drugs include Rv0678, glpK, nuoG, and uvrD1. Genes with variants in the bedaquiline resistant mutants included atpE, fadE28, truA, mmpL5, glnH, and pks8, while clofazimine-resistant mutants displayed ppsD, fbiA, fbiD, mutT3, fadE18, Rv0988, and Rv2082 variants. These results show the importance of epistatic mechanisms as a means of responding to drug pressure and highlight the complexity of resistance acquisition in M. tuberculosis.

Mebendazole Treatment Disrupts the Transcriptional Activity of Hypoxia-Inducible Factors 1 and 2 in Breast Cancer Cells

Breast cancer is the most diagnosed cancer in women in the world. Mebendazole (MBZ) has been demonstrated to have preclinical efficacy across multiple cancers, including glioblastoma multiforme, medulloblastoma, colon, breast, pancreatic, and thyroid cancers. MBZ was also well tolerated in a recent phase I clinical trial of adults diagnosed with glioma. The mechanisms of action reported so far for MBZ include tubulin disruption, inhibiting angiogenesis, promoting apoptosis, and maintaining stemness. To elucidate additional mechanisms of action for mebendazole (MBZ), we performed RNA sequencing of three different breast cancer cell lines treated with either MBZ or vehicle control. We compared the top genes downregulated upon MBZ treatment with expression profiles of cells treated with over 15,000 perturbagens using the clue.io online analysis tool. In addition to tubulin inhibitors, the gene expression profile that correlated most with MBZ treatment matched the profile of cells treated with known hypoxia-inducible factor (HIF-1α and -2α) inhibitors. The HIF pathway is the main driver of the cellular response to hypoxia, which occurs in solid tumors. Preclinical data support using HIF inhibitors in combination with standard of care to treat solid tumors. Therefore, we tested the hypothesis that MBZ could inhibit the hypoxia response. Using RNA sequencing and HIF-reporter assays, we demonstrate that MBZ inhibits the transcriptional activity of HIFs in breast cancer cell lines and in mouse models of breast cancer by preventing the induction of HIF-1α, HIF-2α, and HIF-1β protein under hypoxia. Taken together, our results suggest that MBZ treatment has additional therapeutic efficacy in the setting of hypoxia and warrants further consideration as a cancer therapy.

Past, present and future of Focused Ultrasound as an adjunct or complement to DIPG/DMG therapy: A consensus of the 2021 FUSF DIPG meeting

Diffuse Intrinsic Pontine Glioma (DIPG), now known as Diffuse Midline Glioma (DMG) is a devastating pediatric brain tumor with limited treatment options and a very poor prognosis. Despite more than 250 clinical trials aimed to treat children diagnosed with DMG, no curative therapies currently exist for this patient population. A major obstacle has been the intact blood brain barrier (BBB) which prevents most therapeutics from crossing into the tumor bed. Focused Ultrasound (FUS) is an emerging, noninvasive medical technology which has been shown in both preclinical and clinical research to disrupt the blood brain barrier safely and temporarily. FUS blood brain barrier opening has been studied in combination with chemotherapies in preclinical DMG models, and this technology is now being investigated in clinical trials for the treatment of pediatric brain tumors. Focused ultrasound has additional mechanisms of action, including sonodynamic therapy and radiation sensitization, that hold promise as future DMG therapies as well. This paper, largely based off the proceedings from a workshop held by the Focused Ultrasound Foundation in October of 2021, summarizes the current state of the field of focused ultrasound for DIPG/DMG, including preclinical, technical, and clinical summaries in addition to recommended next steps for continued advancement of the game changing technology of Focused Ultrasound.

Neuronal differentiation drives the antitumor activity of mitogen-activated protein kinase kinase (MEK) inhibition in glioblastoma.

Epidermal growth factor receptor (EGFR) amplification is found in nearly 40%-50% of glioblastoma cases. Several EGFR inhibitors have been tested in glioblastoma but have failed to demonstrate long-term therapeutic benefit, presumably because of acquired resistance. Targeting EGFR downstream signaling with mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) inhibitors would be a more effective approach to glioblastoma treatment. We tested the therapeutic potential of MEK1/2 inhibitors in glioblastoma using 3D cultures of glioma stem-like cells (GSCs) and mouse models of glioblastoma. Several MEK inhibitors were screened in an unbiased high-throughput platform using GSCs. Cell death was evaluated using flow cytometry and Western blotting (WB) analysis. RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and WB analysis were used to identify and validate neuronal differentiation. Unbiased screening of multiple MEK inhibitors in GSCs showed antiproliferative and apoptotic cell death in sensitive cell lines. An RNA-seq analysis of cells treated with trametinib, a potent MEK inhibitor, revealed upregulation of neurogenesis and neuronal differentiation genes, such as achaete-scute homolog 1 (ASCL1), delta-like 3 (DLL3), and neurogenic differentiation 4 (NeuroD4). We validated the neuronal differentiation phenotypes in vitro and in vivo using selected differentiation markers (β-III-tubulin, ASCL1, DLL3, and NeuroD4). Oral treatment with trametinib in an orthotopic GSC xenograft model significantly improved animal survival, with 25%-30% of mice being long-term survivors. Our findings demonstrated that MEK1/2 inhibition promotes neuronal differentiation in glioblastoma, a potential additional mechanism of action of MEK1/2 inhibitors. Thus, MEK inhibitors could be efficacious in glioblastoma patients with activated EGFR/MAPK signaling.

Comparative efficacy and safety of monoamine oxidase type B inhibitors plus channel blockers and monoamine oxidase type B inhibitors as adjuvant therapy to levodopa in the treatment of Parkinson's disease: a network meta-analysis of randomized controlled trials.

The monoamine oxidase type B inhibitors plus channel blockers (MAO-BIs plus) are a new class of antiparkinsonian drug with additional mechanisms of action for their property as ion channel blockers. The present study aimed to compare the efficacy and safety of MAO-BIs plus and conventional MAO-BIs, as well as their corresponding doses, as adjuvant therapy to levodopa in the treatment of Parkinson's disease (PD). Randomized controlled trials enrolling PD patients treated with selegiline, rasagiline, safinamide or zonisamide as adjuvant therapy to levodopa were identified. Bayesian network meta-analysis was conducted. Thirty-one randomized controlled trials comprising 7142 PD patients were included. Compared with levodopa monotherapy, the combination therapy of MAO-BIs and levodopa was significantly more effective, with a mean difference of 2.74 (1.26-4.18) on the Unified Parkinson's Disease Rating Scale (UPDRS) III score change for selegiline, 2.67 (1.45-3.87) for safinamide, 2.2 (0.98-3.64) for zonisamide and 2.04 (1.24-2.87) for rasagiline. No significant difference was detected amongst MAO-BIs. The surface under the cumulative ranking results showed that safinamide 100 mg and rasagiline 1mg ranked first in improving UPDRS III and UPDRS II, respectively. Zonisamide 100 mg ranked first in reducing OFF time. For safety outcomes, rasagiline was associated with a higher incidence of adverse events than placebo and safinamide. MAO-BIs plus had a higher probability of being safer agents compared to conventional MAO-BIs. Monoamine oxidase type B inhibitors plus, conventional MAO-BIs and the corresponding doses are similar in efficacy in PD treatment. MAO-BIs plus might be safer than conventional MAO-BIs. Head-to-head comparisons are needed for further investigation.

Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3.

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet-visible (UV-vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3's lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

What Can Glow Discharge Optical Emission Spectroscopy (GD-OES) Technique Tell Us about Perovskite Solar Cells?

The emerging broad range of applications of the glow discharge optical emission spectroscopy (GD-OES) technique in the field of perovskite solar cells (PSCs) research is reviewed. It can provide a large palette of information by easily and quickly tracking the depth distribution of light to heavy elements. After a discussion of the advantages and the limitations of the technique and a comparison with other analytical techniques, how GD-OES is employed to give structural information on perovskite solar cells is shown. GD-OES has allowed the full perovskite film formation process investigation, from the initial precursor layers containing soaking and complexed solvent to the final crystallized 3D perovskite layers. The A-site elemental cations distribution is followed-up during the film formation. In addition, this technique gives a deep insight into the action mechanism of additives and their effects on the film formation. It provides fruitful information on optimized light absorbing layers and on the selective contact layers which ensure the charge transport in PSCs. It allows to directly visualize halide ions migration and their blocking by ad-hoc chemical engineering and to study the films and PSCs ageing. GD-OES opens new perspectives to explain the final performances of the devices.