Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation.

More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

Using community science to map western monarch butterflies (Danaus plexippus) in spring.

Migratory animals follow seasonal cycles comprising linked phases often with different habitat requirements and demographic processes. Conservation of migratory species therefore must consider the full seasonal cycle to identify points limiting population viability. For western monarch butterflies, which have experienced significant declines, early spring is considered a critical period in the annual population cycle. However, records of western monarchs in early spring, when overall abundance is lowest, have historically been extremely limited. We used a community science initiative, the Western Monarch Mystery Challenge, to collect data on monarch distribution throughout the western United States between February 14th and April 22nd over 3 years. Using data from the Western Monarch Mystery Challenge and iNaturalist, we identified potential breeding habitat for western monarchs in early spring that spanned a large geographic area and several ecoregions. We observed monarchs in early spring that likely eclosed in the current year, suggesting that population expansion from overwintering sites reflects both movement and population growth. The number of records of western monarchs from early spring was higher during the Mystery Challenge (33.0/year) than earlier years (5.1/year). This study demonstrates the potential for and limitations of community science to increase our understanding of species at points in the life cycle when they are rare.

Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation.

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.

Metabolic orchestration of cell death by AMPK-mediated phosphorylation of RIPK1.

Adenosine monophosphate-activated protein kinase (AMPK) activity is stimulated to promote metabolic adaptation upon energy stress. However, sustained metabolic stress may cause cell death. The mechanisms by which AMPK dictates cell death are not fully understood. We report that metabolic stress promoted receptor-interacting protein kinase 1 (RIPK1) activation mediated by TRAIL receptors, whereas AMPK inhibited RIPK1 by phosphorylation at Ser415 to suppress energy stress-induced cell death. Inhibiting pS415-RIPK1 by Ampk deficiency or RIPK1 S415A mutation promoted RIPK1 activation. Furthermore, genetic inactivation of RIPK1 protected against ischemic injury in myeloid Ampkα1-deficient mice. Our studies reveal that AMPK phosphorylation of RIPK1 represents a crucial metabolic checkpoint, which dictates cell fate response to metabolic stress, and highlight a previously unappreciated role for the AMPK-RIPK1 axis in integrating metabolism, cell death, and inflammation.

Making sense of poor adherence in PTSD treatment from the perspectives of veterans and their therapists.

OBJECTIVE: Dropout rates from trauma-focused PTSD treatments (TFTs) in VA clinics are particularly high. We conducted in-depth qualitative interviews with 29 veterans and their therapists to better understand this phenomenon. METHOD: Participants were part of a multisite, mixed-methods study of TFT adherence in VA clinics. Veterans were eligible for interviews if they exhibited poor TFT adherence and screened positive for PTSD in follow-up surveys. Interviews were analyzed using qualitative dyadic analysis approaches. RESULTS: Therapists relied on stereotypes of poor adherence to understand veterans' experiences and were missing information critical to helping veterans succeed. Veterans misunderstood aspects of the therapy and struggled in ways they inadequately expressed to therapists. Therapist attempts at course corrections were poorly matched to veterans' needs. Many dyads reported difficulties in their therapeutic relationships. Veterans reported invalidating experiences that were not prominent in therapists' interviews. CONCLUSIONS: Future work is needed to test hypotheses generated and find effective ways to help veterans fully engage in TFTs. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Impacts of soil nutrition on floral traits, pollinator attraction, and fitness in cucumbers (Cucumis sativus L.).

Annual plants allocate soil nutrients to floral display and pollinator rewards to ensure pollination success in a single season. Nitrogen and phosphorus are critical soil nutrients whose levels are altered by intensive land use that may affect plants' fitness via pollinator attractiveness through floral display and rewards. In a controlled greenhouse study, we studied in cucumbers (Cucumis sativus) how changes in soil nitrogen and phosphorus influence floral traits, including nectar and pollen reward composition. We evaluated how these traits affect bumble bee (Bombus impatiens, an important cucumber pollinator) visitation and ultimately fruit yield. While increasing nitrogen and phosphorus increased growth and floral display, excess nitrogen created an asymptotic or negative effect, which was mitigated by increasing phosphorus. Male floral traits exhibited higher plasticity in responses to changes in soil nutrients than female flowers. At 4:1 nitrogen:phosphorus ratios, male flowers presented increased nectar volume and pollen number resulting in increased bumble bee visitation. Interestingly, other pollinator rewards remained consistent across all soil treatments: male and female nectar sugar composition, female nectar volume, and pollen protein and lipid concentrations. Therefore, although cucumber pollination success was buffered in conditions of nutrient stress, highly skewed nitrogen:phosphorus soil ratios reduced plant fitness via reduced numbers of flowers and reward quantity, pollinator attraction, and ultimately yield.

Can Improved Use of Biomarkers Alter the Fate of PI3K Pathway Inhibitors in the Clinic?

The high frequency of PI3K pathway alterations in cancer has motivated numerous efforts to develop drugs targeting this network. Although many potent and selective inhibitors have been developed and evaluated in preclinical models, their progress to clinical approval has been limited. Here we discuss the pressing need to develop improved biomarker strategies to guide patient selection and improve assessment of patient responses to PI3K pathway inhibitors to address unresolved issues surrounding the efficacy and tolerability of these compounds in patients with cancer.

Positive correlation between transcriptomic stemness and PI3K/AKT/mTOR signaling scores in breast cancer, and a counterintuitive relationship with PIK3CA genotype.

A PI3Kα-selective inhibitor has recently been approved for use in breast tumors harboring mutations in PIK3CA, the gene encoding p110α. Preclinical studies have suggested that the PI3K/AKT/mTOR signaling pathway influences stemness, a dedifferentiation-related cellular phenotype associated with aggressive cancer. However, to date, no direct evidence for such a correlation has been demonstrated in human tumors. In two independent human breast cancer cohorts, encompassing nearly 3,000 tumor samples, transcriptional footprint-based analysis uncovered a positive linear association between transcriptionally-inferred PI3K/AKT/mTOR signaling scores and stemness scores. Unexpectedly, stratification of tumors according to PIK3CA genotype revealed a "biphasic" relationship of mutant PIK3CA allele dosage with these scores. Relative to tumor samples without PIK3CA mutations, the presence of a single copy of a hotspot PIK3CA variant was associated with lower PI3K/AKT/mTOR signaling and stemness scores, whereas the presence of multiple copies of PIK3CA hotspot mutations correlated with higher PI3K/AKT/mTOR signaling and stemness scores. This observation was recapitulated in a human cell model of heterozygous and homozygous PIK3CAH1047R expression. Collectively, our analysis (1) provides evidence for a signaling strength-dependent PI3K-stemness relationship in human breast cancer; (2) supports evaluation of the potential benefit of patient stratification based on a combination of conventional PI3K pathway genetic information with transcriptomic indices of PI3K signaling activation.

Veterans with poor PTSD treatment adherence: Exploring their loved ones' experience of PTSD and understanding of PTSD treatment.

Trauma-focused psychotherapies such as cognitive processing therapy (CPT) and prolonged exposure (PE) are some of the most effective treatments available for posttraumatic stress disorder (PTSD). These treatments have been widely disseminated and promoted throughout the VA Health care System. However, adherence to and completion of these protocols among veterans is often poor, resulting in diminished impact. "Support persons" (SPs) such as relatives and close friends may provide a source of emotional or practical support in treatment, but little is known about how SPs are involved in or exposed to treatment principles and activities. The primary goal of the current research was to examine the experience of SPs of veterans who had poor adherence to treatment. We were interested in SPs' knowledge about the treatment, their level of involvement in treatment activities or sessions, and their potential interest in more participation or education. Qualitative analyses were used to examine data collected from interviews with 19 SPs of veterans who had an unsuccessful course of CPT or PE. Results indicated generally very low levels of knowledge and treatment participation. However, among most SPs there was substantial interest in the possibility of more treatment involvement, particularly in order to receive guidance from the clinician about how to respond to the veteran's symptoms. We suggest that it is possible and desirable to incorporate loved ones more formally into such protocols. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

A Theory of Planned Behavior Scale for Adherence to Trauma-Focused Posttraumatic Stress Disorder Treatments.

Evidence-based psychotherapies for posttraumatic stress disorder (PTSD), such as cognitive processing therapy and prolonged exposure (CPT/PE), greatly reduce suffering for veterans, but many veterans fail to complete treatment. Developing a theory-based understanding of adherence is necessary to inform interventions to improve treatment retention. We developed and tested a series of scales applying the theory of planned behavior (TPB) to CPT/PE adherence. The scales were administered in mailed surveys as part of a larger mixed-methods study of veteran adherence to PE/CPT. Surveys were sent to 379 veterans who were initiating CPT/PE across four U.S. Veterans Affairs (VA) hospitals and 207 of their loved ones. Subsequent session attendance and homework compliance were coded via a review of electronic medical records. We examined item-level characteristics, factor structure, and the convergent and discriminant validity of the resultant scales. The findings support four subscales: two related to attitudes (i.e., Treatment Makes Sense and Treatment Fits Needs), one related to perceived behavioral control over participation (i.e., Participation Control), and one related to perceived family attitudes about CPT/PE participation (i.e., Subjective Norms). Scale validity was supported through significant associations with theoretically relevant constructs, including intentions to persist in CPT/PE, rs = .19-.38; treatment completion, rs = .21-.25; practical treatment barriers, rs = -.19 to -.24; and therapeutic alliance, rs = .39-.57.

Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling.

The PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over half of tumors exhibiting aberrant AKT activation. Although potent small-molecule AKT inhibitors have entered clinical trials, robust and durable therapeutic responses have not been observed. As an alternative strategy to target AKT, we report the development of INY-03-041, a pan-AKT degrader consisting of the ATP-competitive AKT inhibitor GDC-0068 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon (CRBN). INY-03-041 induced potent degradation of all three AKT isoforms and displayed enhanced anti-proliferative effects relative to GDC-0068. Notably, INY-03-041 promoted sustained AKT degradation and inhibition of downstream signaling effects for up to 96 h, even after compound washout. Our findings suggest that AKT degradation may confer prolonged pharmacological effects compared with inhibition, and highlight the potential advantages of AKT-targeted degradation.

CLOCK regulates mammary epithelial cell growth and differentiation.

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein (P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates (P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects (P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63 qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively (P < 0.05), in shClock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with shClock These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.

Tissue-specific changes in molecular clocks during the transition from pregnancy to lactation in mice.

Circadian clocks regulate homeostasis and mediate responses to stressors. Lactation is one of the most energetically demanding periods of an adult female's life. Peripartum changes occur in almost every organ so the dam can support neonatal growth through milk production while homeostasis is maintained. How circadian clocks are involved in adaptation to lactation is currently unknown. The abundance and temporal pattern of core clock genes' expression were measured in suprachiasmatic nucleus, liver, and mammary from late pregnant and early lactation mice. Tissue-specific changes in molecular clocks occurred between physiological states. Amplitude and robustness of rhythms increased in suprachiasmatic nucleus and liver. Mammary rhythms of core molecular clock genes were suppressed. Attenuated rhythms appeared to be a physiological adaptation of mammary to lactation, because manipulation of timing of suckling resulting in significant differences in plasma prolactin and corticosterone had no effect on amplitude. Analysis of core clock proteins revealed that the stoichiometric relationship between positive (CLOCK) and negative (PER2) components remained 1:1 in liver but was increased to 4:1 in mammary during physiological transition. Induction of differentiation of mammary epithelial cell line HC11 with dexamethasone, insulin, and prolactin resulted in similar stoichiometric changes among positive and negative clock regulators, and prolactin induced phase shifts in HC11 Arntl expression rhythm. Data support that distinct mechanisms drive periparturient changes in mammary clock. Stoichiometric change in clock regulators occurs with gland differentiation. Suppression of mammary clock gene expression rhythms represents a physiological adaptation to suckling cues. Adaptations in mammary clock are likely needed in part to support suckling demands of neonates.