Eganelisib combined with immune checkpoint inhibitor therapy and chemotherapy in frontline metastatic triple-negative breast cancer triggers macrophage reprogramming, immune activation and extracellular matrix reorganization in the tumor microenvironment.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis particularly in the metastatic setting. Treatments with anti-programmed cell death protein-1/programmed death-ligand 1 (PD-L1) immune checkpoint inhibitors (ICI) in combination with chemotherapies have demonstrated promising clinical benefit in metastatic TNBC (mTNBC) but there is still an unmet need, particularly for patients with PD-L1 negative tumors. Mechanisms of resistance to ICIs in mTNBC include the presence of immunosuppressive tumor-associated macrophages (TAMs) in the tumor microenvironment (TME). Eganelisib is a potent and selective, small molecule PI3K-γ inhibitor that was shown in preclinical studies to reshape the TME by reducing myeloid cell recruitment to tumors and reprogramming TAMs from an immune-suppressive to an immune-activating phenotype and enhancing activity of ICIs. These studies provided rationale for the clinical evaluation of eganelisib in combination with the anti-PD-L1 atezolizumab and nab-paclitaxel in firstline mTNBC in the phase 2 clinical trial MAcrophage Reprogramming in Immuno-Oncology-3 (MARIO-3, NCT03961698). We present here for the first time, in-depth translational analyses from the MARIO-3 study and supplemental data from eganelisib monotherapy Ph1/b study in solid tumors (MARIO-1, NCT02637531). METHODS: Paired pre-treatment and post-treatment tumor biopsies were analyzed for immunophenotyping by multiplex immunofluorescence (n=11), spatial transcriptomics using GeoMx digital spatial profiling (n=12), and PD-L1 immunohistochemistry, (n=18). Peripheral blood samples were analyzed using flow cytometry and multiplex cytokine analysis. RESULTS: Results from paired tumor biopsies from MARIO-3 revealed gene signatures of TAM reprogramming, immune activation and extracellular matrix (ECM) reorganization. Analysis of PD-L1 negative tumors revealed elevated ECM gene signatures at baseline that decreased after treatment. Gene signatures of immune activation were observed regardless of baseline PD-L1 status and occurred in patients having longer progression-free survival. Peripheral blood analyses revealed systemic immune activation. CONCLUSIONS: This is the first report of translational analyses including paired tumor biopsies from a phase 2 clinical study of the first-in-class PI3K-γ inhibitor eganelisib in combination with atezolizumab and nab-paclitaxel in frontline mTNBC. These results support the mechanism of action of eganelisib as a TAM-reprogramming immunotherapy and support the rationale for combining eganelisib with ICI and chemotherapy in indications with TAM-driven resistance to ICI.

PI3Kγ inhibition circumvents inflammation and vascular leak in SARS-CoV-2 and other infections.

Virulent infectious agents such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and methicillin-resistant Staphylococcus aureus (MRSA) induce tissue damage that recruits neutrophils, monocyte, and macrophages, leading to T cell exhaustion, fibrosis, vascular leak, epithelial cell depletion, and fatal organ damage. Neutrophils, monocytes, and macrophages recruited to pathogen-infected lungs, including SARS-CoV-2-infected lungs, express phosphatidylinositol 3-kinase gamma (PI3Kγ), a signaling protein that coordinates both granulocyte and monocyte trafficking to diseased tissues and immune-suppressive, profibrotic transcription in myeloid cells. PI3Kγ deletion and inhibition with the clinical PI3Kγ inhibitor eganelisib promoted survival in models of infectious diseases, including SARS-CoV-2 and MRSA, by suppressing inflammation, vascular leak, organ damage, and cytokine storm. These results demonstrate essential roles for PI3Kγ in inflammatory lung disease and support the potential use of PI3Kγ inhibitors to suppress inflammation in severe infectious diseases.

Heart rate reactivity mediates the relationship between trait gratitude and acute myocardial infarction.

OBJECTIVE: This study examines the relationship between trait gratitude and acute myocardial infarction. A burgeoning body of literature suggests that gratitude can play a role in regulating individual's cardiovascular responses to stress which in turn, may reduce the incidence of cardiovascular disease such as acute myocardial infarction. However, to date no research has examined these effects. METHOD: This study used the Mid-Life in the United States dataset (MIDUS; N = 1031) to assess these relationships. Participants completed a standardised cardiovascular stress-testing laboratory protocol and were assessed at a second time-point; on average 6.7 years later. RESULTS: Results from logistic parallel mediation models suggest that trait gratitude was found to be significantly associated with reduced risk of acute myocardial infarction through the mechanism of increased heart rate reactivity, ß = -0.098, 95%CI [- 0.331, - 0.010]. However, neither systolic nor diastolic blood pressure reactivity mediated this relationship. CONCLUSIONS: These findings suggest that gratitude may be associated with certain aspects of physical health. Specifically, our study reveals a potential link between gratitude and cardiovascular reactivity, which could be a mechanism through which trait gratitude contributes to reductions in the risk of myocardial infarction. As such, this study highlights the potential utility of positive psychological factors, such as gratitude, in promoting cardiovascular health.

Eganelisib, a First-in-Class PI3Kγ Inhibitor, in Patients with Advanced Solid Tumors: Results of the Phase 1/1b MARIO-1 Trial.

PURPOSE: Eganelisib (IPI-549) is a first-in-class, orally administered, highly selective PI3Kγ inhibitor with antitumor activity alone and in combination with programmed cell death protein 1/ligand 1 (PD-1/PD-L1) inhibitors in preclinical studies. This phase 1/1b first-in-human, MAcrophage Reprogramming in Immuno-Oncology-1 (NCT02637531) study evaluated the safety and tolerability of once-daily eganelisib as monotherapy and in combination with nivolumab in patients with solid tumors. PATIENTS AND METHODS: Dose-escalation cohorts received eganelisib 10-60 mg as monotherapy (n = 39) and 20-40 mg when combined with nivolumab (n = 180). Primary endpoints included incidence of dose-limiting toxicities (DLT) and adverse events (AE). RESULTS: The most common treatment-related grade ≥3 toxicities with monotherapy were increased alanine aminotransferase (ALT; 18%), aspartate aminotransferase (AST; 18%), and alkaline phosphatase (5%). No DLTs occurred in the first 28 days; however, toxicities meeting DLT criteria (mostly grade 3 reversible hepatic enzyme elevations) occurred with eganelisib 60 mg in later treatment cycles. In combination, the most common treatment-related grade ≥3 toxicities were increased AST (13%) and increased ALT and rash (10%). Treatment-related serious AEs occurred in 5% of monotherapy patients (grade 4 bilirubin and hepatic enzyme increases in one patient each) and 13% in combination (pyrexia, rash, cytokine release syndrome, and infusion-related reaction in ≥2 patients each). Antitumor activity was observed in combination, including patients who had progressed on PD-1/PD-L1 inhibitors. CONCLUSIONS: On the basis of the observed safety profile, eganelisib doses of 30 and 40 mg once daily in combination with PD-1/PD-L1 inhibitors were chosen for phase 2 study.

Gratitude, affect balance, and stress buffering: A growth curve examination of cardiovascular responses to a laboratory stress task.

Previous research has indicated that gratitude and affect-balance play key stress-buffering roles. However, to date there is limited research on the impact of gratitude and affect balance on cardiovascular recovery from acute psychological stress, and whether affect balance moderates the relationship between gratitude and cardiovascular reactions to acute psychological stress. In this study, 68 adults completed measures of state gratitude, positive and negative affect, and completed a laboratory-based cardiovascular stress-testing protocol. This incorporated a 20-minute acclimatization period, a 10-minute baseline, a 6-minute arithmetic stress task, and an 8-minute recovery period. Mixed-effects growth curve models were fit and the results indicated that state gratitude predicted lower systolic blood pressure responses throughout the stress-testing period. Affect balance was found to moderate the association between state gratitude and diastolic blood pressure responses to stress, amplifying the effects of state gratitude. These findings suggest that state gratitude has a unique stress-buffering effect on both reactions to and recovery from acute psychological stress.

Health literacy among self-help leprosy group members reduces stereotype endorsement and stigma-related harm in rural Nepal.

There is increasing appreciation that group memberships can have both beneficial and damaging impacts on health. In collaboration with Nepal Leprosy Trust (NLT), this longitudinal study explores a group-based approach to stigma reduction among people affected by leprosy in rural Nepal (N = 71)-a hard to reach and underrepresented non-WEIRD population. Informed by the 'social cure' literature, and the progressive model of self-stigma, we use a longitudinal design. We found that a sense of belonging to a self-help group can facilitate education in terms of health literacy, and over time these two factors also have impacts on participants stigma. Specifically, self-help group belonging predicted improvements in health literacy, leading to reduced endorsement of negative stereotypes and thus less stigma-related harm among people affected by leprosy. The study offers promising evidence that group-based interventions, which support health education, can reduce the harmful impact of stigma in very challenging contexts.

Building Resources in Caregivers: Feasibility of a Brief Writing Intervention to Increase Benefit Finding in Caregivers.

The Building Resources in Caregivers (BRiC) is a pilot feasibility trial that compared the effects of a 2-week benefit finding writing expressive intervention to a control intervention, who wrote about the weather. Caregivers completed primary (benefit finding) and secondary (quality of life, depression and anxiety) outcome measures at pre (t1), immediately post-test (t2) and 1 month later (t3). They also completed measures relating to trial feasibility, difficulty, and acceptance. Using complete case analysis only, analysis revealed no effect of the intervention for primary or secondary outcomes. Despite this, there were no differences between the intervention and control groups on key feasibility measures. Caregivers in the control condition were less likely to recommend this to other caregivers. Moreover, qualitative commentary provided by caregivers suggested that not everyone enjoyed the writing, some found it stressful, offering up some explanation for our findings. Our pilot trial suggests that any future benefit-finding writing intervention would require several procedure modifications including tailoring to a specific cohort of caregivers, in particular those who like writing, before it has some utility as a psychosocial intervention.

Psychosocial health mediates the gratitude-physical health link.

There is now a growing body of research demonstrating the physical health benefits of being grateful. However, research has only just began to explore the mechanisms accounting for this gratitude-health relationship. This study examines the relationship between dispositional gratitude and self-reported physical health symptoms, and explores whether this relationship is explained through reduced levels of perceived loneliness and stress. This study employed a cross-sectional design with a sample of 607 healthy adults. Serial mediation analysis revealed that the positive effect of gratitude on physical health was significantly mediated by lower reported levels of perceived loneliness and stress. These findings are important given evidence that gratitude can be cultivated, and may serve to buffer against stress and loneliness and improve somatic health symptoms in the general population.

Feeling Thanks and Saying Thanks: A Randomized Controlled Trial Examining If and How Socially Oriented Gratitude Journals Work.

OBJECTIVE: This study examined the effect of a reflective interpersonal gratitude journal, a reflective-behavioral interpersonal gratitude journal and an active control journal, on primary qualities of well-being and depression. METHOD: Participants (n = 192; 67.2% female) completed this 3-month longitudinal randomized controlled design. RESULTS: Participants in the reflective-behavioral condition experienced the greatest improvements in affect balance and reductions in depression at immediate posttest. Both gratitude interventions improved affect balance at 1 month, compared to the control. Changes in affect balance for those in the reflective-behavioral condition were mediated by the rate at which people expressed gratitude in their existing relationships. This effect was moderated by participant's baseline depressive status. CONCLUSION: Expressing felt gratitude to others appears to be a crucial step in deriving benefits, and these benefits may not be limited to the emotionally healthy. Given the applied popularity of gratitude interventions, understanding not only if but also how they work is essential.

A randomised controlled trial of benefit finding in caregivers: The Building Resources in Caregivers Study Protocol.

Caregivers may engage in benefit finding, that is, an increase in perceived positive growth, as a cognitive strategy for coping with stress. The Building Resources in Caregivers study will compare effects of a brief benefit finding writing intervention with a control intervention. Caregivers of people with mental and physical disabilities will be randomised into either a benefit-writing group or a neutral writing group. Caregivers will complete measures relating to themselves and care-recipients (e.g. sociodemographics and illness type) and psychometric measures of benefit finding, distress and quality of life at three time points. Additionally, qualitative commentary on participation experiences will be gathered.

HSP90 inhibition enhances antimitotic drug-induced mitotic arrest and cell death in preclinical models of non-small cell lung cancer.

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.

An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.

The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7(Fbxw8) localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7(Fbxw8) by independent approaches including Fbxw8 knockdown reveals that Cul7(Fbxw8) is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7(Fbxw8) also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7(Fbxw8) in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8) in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8) ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.

A genome-wide camptothecin sensitivity screen identifies a mammalian MMS22L-NFKBIL2 complex required for genomic stability.

Replication stress involving collision of replisomes with camptothecin (CPT)-stabilized DNA-Topoisomerase I adducts activates an ATR-dependent pathway to promote repair by homologous recombination. To identify human genes that protect cells from such replication stress, we performed a genome-wide CPT sensitivity screen. Among numerous candidate genes are two previously unstudied proteins: the ankyrin repeat protein NFKBIL2 and C6ORF167 (MMS22L), distantly related to yeast replication stress regulator Mms22p. MMS22L and NFKBIL2 interact with each other and with FACT (facilitator of chromatin transcription) and MCM (minichromosome maintenance) complexes. Cells depleted of NFKBIL2 or MMS22L are sensitive to DNA-damaging agents, load phosphorylated RPA onto chromatin in a CTIP-dependent manner, activate the ATR/ATRIP-CHK1 and double-strand break repair signaling pathways, and are defective in HR. This study identifies MMS22L-NFKBIL2 as components of the replication stress control pathway and provides a resource for discovery of additional components of this pathway.

Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.

Structure-specific endonucleases mediate cleavage of DNA structures formed during repair of collapsed replication forks and double-strand breaks (DSBs). Here, we identify BTBD12 as the human ortholog of the budding yeast DNA repair factor Slx4p and D. melanogaster MUS312. Human SLX4 forms a multiprotein complex with the ERCC4(XPF)-ERCC1, MUS81-EME1, and SLX1 endonucleases and also associates with MSH2/MSH3 mismatch repair complex, telomere binding complex TERF2(TRF2)-TERF2IP(RAP1), the protein kinase PLK1 and the uncharacterized protein C20orf94. Depletion of SLX4 causes sensitivity to mitomycin C and camptothecin and reduces the efficiency of DSB repair in vivo. SLX4 complexes cleave 3' flap, 5' flap, and replication fork structures; yet unlike other endonucleases associated with SLX4, the SLX1-SLX4 module promotes symmetrical cleavage of static and migrating Holliday junctions (HJs), identifying SLX1-SLX4 as a HJ resolvase. Thus, SLX4 assembles a modular toolkit for repair of specific types of DNA lesions and is critical for cellular responses to replication fork failure.

Myc inhibits p27-induced erythroid differentiation of leukemia cells by repressing erythroid master genes without reversing p27-mediated cell cycle arrest.

Inhibition of differentiation has been proposed as an important mechanism for Myc-induced tumorigenesis, but the mechanisms involved are unclear. We have established a genetically defined differentiation model in human leukemia K562 cells by conditional expression of the cyclin-dependent kinase (Cdk) inhibitor p27 (inducible by Zn(2+)) and Myc (activatable by 4-hydroxy-tamoxifen). Induction of p27 resulted in erythroid differentiation, accompanied by Cdk inhibition and G(1) arrest. Interestingly, activation of Myc inhibited p27-mediated erythroid differentiation without affecting p27-mediated proliferation arrest. Microarray-based gene expression indicated that, in the presence of p27, Myc blocked the upregulation of several erythroid-cell-specific genes, including NFE2, JUNB, and GATA1 (transcription factors with a pivotal role in erythropoiesis). Moreover, Myc also blocked the upregulation of Mad1, a transcriptional antagonist of Myc that is able to induce erythroid differentiation. Cotransfection experiments demonstrated that Myc-mediated inhibition of differentiation is partly dependent on the repression of Mad1 and GATA1. In conclusion, this model demonstrates that Myc-mediated inhibition of differentiation depends on the regulation of a specific gene program, whereas it is independent of p27-mediated cell cycle arrest. Our results support the hypothesis that differentiation inhibition is an important Myc tumorigenic mechanism that is independent of cell proliferation.

Ubiquitin proteasome system (UPS): what can chromatin do for you?

Cul4-Ddb1, a RING H2 ubiquitin ligase, plays an important role in many vital cellular processes including DNA replication, DNA repair and transcription. Recent research reveals strong links between Cul4-mediated signaling pathways and chromatin biology. Ubiquitylation of substrates by Cul4-Ddb1 occurs on chromatin and is initiated by chromatin-based signals that either recruit Cul4-Ddb1 to chromatin or alter the activity of the ligase. This includes Cul4-mediated ubiquitylation of the replication licensing factor, Cdt1; a process that requires chromatin-bound PCNA and Cdt2, a member of the recently identified family of candidate substrate receptors for Cul4 (termed Ddb1- and Cul4-associated factors: DCAFs). The activity of two other Cul4-based ubiquitin ligases, Cul4-Ddb1(Ddb2) and Cul4-Ddb1(CSA), are differentially regulated by the COP9 signalosome in response to different chromatin-based signals. Finally, examples of direct modifications to chromatin by Cul4-Ddb1 have emerged, including ubiquitylation of histones and the recruitment of enzymes involved in chromatin remodeling or histone methylation.

c-Myc localization within the nucleus: evidence for association with the PML nuclear body.

Definitive localization of c-Myc within the nucleus is important to fully understand the regulation and function of this oncoprotein. Studies of c-Myc distribution, however, have produced conflicting results. To overcome technical challenges inherent in c-Myc cytology, we use here three methods to visualize c-Myc and in addition examine the impact of proteasome inhibition. EYFP or HA-tagged Myc was reintroduced by stable transfection into myc null diploid rat fibroblasts, replacing endogenous Myc with tagged Myc expressed at or near normal levels. This tagged Myc is shown to functionally replace the endogenous Myc by restoration of normal cell morphology and growth rate. We were able to confirm key findings using antibodies to the endogenous c-Myc and/or its partner, Max. Contrary to some published reports, by all three methods the c-Myc protein in rat fibroblasts distributes predominantly throughout the nucleus in a dispersed granular pattern, avoiding the nucleolus. Importantly, however, several findings provide evidence for an unanticipated relationship between c-Myc and PML nuclear bodies, which is enhanced under conditions of proteasome inhibition. Evidence of Max concentration within PML bodies is shown both with and without proteasome inhibition, strengthening the relationship between PML bodies and Myc/Max. Some accumulation of Myc and Max in nucleoli upon proteasome inhibition is also observed, although co-localization of ubiquitin was only seen with PML bodies. This work provides a comprehensive study of c-Myc distribution and also presents the first evidence of a relationship between turnover of this oncoprotein and PML nuclear bodies, known to break down in certain cancers.

A large scale genetic analysis of c-Myc-regulated gene expression patterns.

The myc proto-oncogenes encode transcriptional regulators whose inappropriate expression is correlated with a wide array of human malignancies. Up-regulation of Myc enforces growth, antagonizes cell cycle withdrawal and differentiation, and in some situations promotes apoptosis. How these phenotypes are elicited is not well understood, largely because we lack a clear picture of the biologically relevant downstream effectors. We created a new biological system for the optimal profiling of Myc target genes based on a set of isogenic c-myc knockout and conditional cell lines. The ability to modulate Myc activity from essentially null to supraphysiological resulted in a significantly increased and reproducible yield of targets and revealed a large subset of genes that respond optimally to Myc in its physiological range of expression. The total extent of transcriptional changes that can be triggered by Myc is remarkable and involves thousands of genes. Although the majority of these effects are not direct, many of the indirect targets are likely to have important roles in mediating the elicited cellular phenotypes. Myc-activated functions are indicative of a physiological state geared toward the rapid utilization of carbon sources, the biosynthesis of precursors for macromolecular synthesis, and the accumulation of cellular mass. In contrast, the majority of Myc-repressed genes are involved in the interaction and communication of cells with their external environment, and several are known to possess antiproliferative or antimetastatic properties.

A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism.

A cDNA library enriched with Myc-responsive cDNAs but depleted of myc cDNAs was used in a functional screen for growth enhancement in c-myc-null cells. A cDNA clone for mitochondrial serine hydroxymethyltransferase (mSHMT) that was capable of partial complementation of the growth defects of c-myc-null cells was identified. Expression analysis and chromatin immunoprecipitation demonstrated that mSHMT is a direct Myc target gene. Furthermore, a separate gene encoding the cytoplasmic isoform of the same enzyme is also a direct target of Myc regulation. SHMT enzymes are the major source of the one-carbon unit required for folate metabolism and for the biosynthesis of nucleotides and amino acids. Our data establish a novel functional link between Myc and the regulation of cellular metabolism.