Integrative modeling uncovers p21-driven drug resistance and prioritizes therapies for PIK3CA-mutant breast cancer.

Utility of PI3Kα inhibitors like BYL719 is limited by the acquisition of genetic and non-genetic mechanisms of resistance which cause disease recurrence. Several combination therapies based on PI3K inhibition have been proposed, but a way to systematically prioritize them for breast cancer treatment is still missing. By integrating published and in-house studies, we have developed in silico models that quantitatively capture dynamics of PI3K signaling at the network-level under a BYL719-sensitive versus BYL719 resistant-cell state. Computational predictions show that signal rewiring to alternative components of the PI3K pathway promote resistance to BYL719 and identify PDK1 as the most effective co-target with PI3Kα rescuing sensitivity of resistant cells to BYL719. To explore whether PI3K pathway-independent mechanisms further contribute to BYL719 resistance, we performed phosphoproteomics and found that selection of high levels of the cell cycle regulator p21 unexpectedly promoted drug resistance in T47D cells. Functionally, high p21 levels favored repair of BYL719-induced DNA damage and bypass of the associated cellular senescence. Importantly, targeted inhibition of the check-point inhibitor CHK1 with MK-8776 effectively caused death of p21-high T47D cells, thus establishing a new vulnerability of BYL719-resistant breast cancer cells. Together, our integrated studies uncover hidden molecular mediators causing resistance to PI3Kα inhibition and provide a framework to prioritize combination therapies for PI3K-mutant breast cancer.

Post-COVID-19 vaccination myocarditis: a prospective cohort study pre and post vaccination using cardiovascular magnetic resonance.

BACKGROUND: Concerns about COVID-19 vaccination induced myocarditis or subclinical myocarditis persists in some populations. Cardiac magnetic resonance imaging (CMR) has been used to detect signs of COVID-19 vaccination induced myocarditis. This study aims to: (i) characterise myocardial tissue, function, size before and after COVID-19 vaccination, (ii) determine if there is imaging evidence of subclinical myocardial inflammation or injury after vaccination using CMR. METHODS: Subjects aged ≥ 12yrs old without prior COVID-19 or COVID-19 vaccination underwent two CMR examinations: first, ≤ 14 days before the first COVID-19 vaccination and a second time ≤ 14 days after the second COVID-19 vaccination. Biventricular indices, ejection fraction (EF), global longitudinal strain (GLS), late gadolinium enhancement (LGE), left ventricular (LV) myocardial native T1, T2, extracellular volume (ECV) quantification, lactate dehydrogenase (LDH), white cell count (WCC), C-reactive protein (CRP), NT-proBNP, troponin-T, electrocardiogram (ECG), and 6-min walk test were assessed in a blinded fashion. RESULTS: 67 subjects were included. First and second CMR examinations were performed a median of 4 days before the first vaccination (interquartile range 1-8 days) and 5 days (interquartile range 3-6 days) after the second vaccination respectively. No significant change in global native T1, T2, ECV, LV EF, right ventricular EF, LV GLS, LGE, ECG, LDH, troponin-T and 6-min walk test was demonstrated after COVID-19 vaccination. There was a significant WCC decrease (6.51 ± 1.49 vs 5.98 ± 1.65, p = 0.003) and CRP increase (0.40 ± 0.22 vs 0.50 ± 0.29, p = 0.004). CONCLUSION: This study found no imaging, biochemical or ECG evidence of myocardial injury or inflammation post COVID-19 vaccination, thus providing some reassurance that COVID-19 vaccinations do not typically cause subclinical myocarditis.

A decade of progress in juvenile idiopathic athritis treatments and outcomes in Canada: results from ReACCh-Out and the CAPRI registry.

OBJECTIVE: To assess changes in juvenile idiopathic arthritis (JIA) treatments and outcomes in Canada, comparing a 2005-2010 and a 2017-2021 inception cohorts. METHODS: Patients enrolled within three months of diagnosis in the Research in Arthritis in Canadian Children Emphasizing Outcomes (ReACCh-Out) and the Canadian Alliance of Pediatric Rheumatology Investigators Registry (CAPRI) cohorts were included. Cumulative incidences of drug starts and outcome attainment within 70 weeks of diagnosis were compared with Kaplan Meier survival analysis and multivariable Cox regression. RESULTS: The 2005-2010 and 2017-2021 cohorts included 1128 and 721 patients, respectively. JIA category distribution and baseline clinical juvenile idiopathic arthritis disease activity (cJADAS10) scores at enrolment were comparable. By 70 weeks, 6% of patients (95% CI 5, 7) in the 2005-2010 and 26% (23, 30) in the 2017-2021 cohort had started a biologic DMARD (bDMARD), and 43% (40, 47) and 60% (56, 64) had started a conventional DMARD (cDMARD), respectively. Outcome attainment was 64% (61, 67) and 83% (80, 86) for Inactive disease (Wallace criteria), 69% (66, 72) and 84% (81, 87) for minimally active disease (cJADAS10 criteria), 57% (54, 61) and 63% (59, 68) for pain control (<1/10), and 52% (47, 56) and 54% (48, 60) for a good health-related quality of life. CONCLUSION: Although baseline disease characteristics were comparable in the 2005-2010 and 2017-2021 cohorts, cDMARD and bDMARD use increased with a concurrent increase in minimally active and inactive disease. Improvements in parent and patient reported outcomes were smaller than improvements in disease activity.

Canadian Rheumatology Association Recommendations for the Screening, Monitoring, and Treatment of Juvenile Idiopathic Arthritis-Associated Uveitis.

OBJECTIVE: To develop Canadian recommendations for the screening, monitoring, and treatment of uveitis associated with juvenile idiopathic arthritis (JIA). METHODS: Recommendations were developed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE)-ADOLOPMENT approach. A working group of 14 pediatric rheumatologists, 6 ophthalmologists, 2 methodologists, and 3 caregiver/patient representatives reviewed recent American College of Rheumatology (ACR)/Arthritis Foundation (AF) recommendations and worked in pairs to develop evidence-to-decision (EtD) tables. A survey to assess agreement and recommendations requiring group discussion was completed. EtD tables were presented, discussed, and voted upon at a virtual meeting, to produce the final recommendations. A health equity framework was applied to all aspects of the adolopment process including the EtD tables, survey responses, and virtual meeting discussion. RESULTS: The survey identified that 7 of the 19 recommendations required rigorous discussion. Seventy-five percent of working group members attended the virtual meeting to discuss controversial topics as they pertained to the Canadian environment, including timing to first eye exam, frequency of screening, escalation criteria for systemic and biologic therapy, and the role of nonbiologic therapies. Equity issues related to access to care and advanced therapeutics across Canadian provinces and territories were highlighted. Following the virtual meeting, 5 recommendations were adapted, 2 recommendations were removed, and 1 was developed de novo. CONCLUSION: Recommendations for JIA-associated uveitis were adapted to the Canadian context by a working group of pediatric rheumatologists, ophthalmologists with expertise in the management of uveitis, and parent/patient input, taking into consideration cost, equity, and access.

Generation and functional characterization of murine mammary organoids.

3D cultures of mammary epithelial cells purified from murine models provide a unique resource to study genetically defined breast cancer and response to targeted therapies. Here, we describe step-by-step experimental procedures for the successful establishment of murine mammary organoid lines isolated from mammary glands or mammary tumors driven by mutations in components of the PI3K pathway. These detailed protocols also include procedures to perform assays that can be adopted to screen response to drug treatments and to inform better therapies. For details on potential applications and use of this protocol, please refer to Yip et al. (2020).

Chronic recurrent multifocal osteomyelitis and primary sclerosing cholangitis with type 1 autoimmune hepatitis in a child with ulcerative colitis: a case report.

BACKGROUND: Chronic Recurrent Multifocal Osteomyelitis (CRMO) is a condition characterized by sterile bone inflammation, usually occurring in childhood. Although the etiology remains unclear, this condition has been associated with inflammatory bowel disease (IBD). Primary sclerosing cholangitis (PSC) and Autoimmune Hepatitis (AIH) are also uncommon pediatric conditions with a known association with IBD. CASE PRESENTATION: We present a unique case of a pediatric patient with an initial diagnosis of CRMO, with subsequent diagnosis of autoimmune hepatitis and PSC overlap, and eventually IBD. CONCLUSIONS: Patients with CRMO may also develop PSC in addition to IBD, further highlighting the importance of IBD pathophysiology in both conditions. Clinical screening of associated gastrointestinal findings may be of value in patients with CRMO.

Signaling Pathways in Cancer: Therapeutic Targets, Combinatorial Treatments, and New Developments.

Molecular alterations in cancer genes and associated signaling pathways are used to inform new treatments for precision medicine in cancer. Small molecule inhibitors and monoclonal antibodies directed at relevant cancer-related proteins have been instrumental in delivering successful treatments of some blood malignancies (e.g., imatinib with chronic myelogenous leukemia (CML)) and solid tumors (e.g., tamoxifen with ER positive breast cancer and trastuzumab for HER2-positive breast cancer). However, inherent limitations such as drug toxicity, as well as acquisition of de novo or acquired mechanisms of resistance, still cause treatment failure. Here we provide an up-to-date review of the successes and limitations of current targeted therapies for cancer treatment and highlight how recent technological advances have provided a new level of understanding of the molecular complexity underpinning resistance to cancer therapies. We also raise three basic questions concerning cancer drug discovery based on molecular markers and alterations of selected signaling pathways, and further discuss how combination therapies may become the preferable approach over monotherapy for cancer treatments. Finally, we consider novel therapeutic developments that may complement drug delivery and significantly improve clinical response and outcomes of cancer patients.

The validity, reliability, sensitivity and responsiveness of a modified Patient Enablement Instrument (PEI-2) as a tool for serial measurements of health enablement.

BACKGROUND: Patient enablement is a core tenet of patient-centred and holistic primary care. The Patient Enablement Instrument (PEI) is a transitional measure limited in its ability to measure changes over time. A modified version, PEI-2, has been developed to measure enablement at a given time-point without comparison to a recalled baseline. OBJECTIVE: To assess the validity, reliability, sensitivity and responsiveness of PEI-2. METHODS: PEI-2 was modified from the Chinese PEI to assess enablement over 4 weeks in a prospective cohort study nested within a community support programme [Trekkers Family Enhancement Scheme (TFES)] in Hong Kong. Construct validity was assessed by factor analysis and convergent validity by Spearman's correlations with health-related quality of life and depressive symptoms. Internal reliability was assessed using Cronbach's alpha. Test-retest reliability was assessed by intraclass correlation (ICC), responsiveness by 12-24-month change in PEI-2 score and sensitivity by differences in change of PEI-2 score between TFES participants and a control group. RESULTS: PEI-2 demonstrated construct validity with all items loading on one factor (factor loadings >0.7). Convergent validity was confirmed by significant correlations with 12-item Short Form Questionnaire, version 2 (r = 0.1089-0.1919) and Patient Health Questionnaire-9 (r = -0.2030). Internal reliability was high (Cronbach's alpha = 0.9095) and test-retest reliability moderate (ICC = 0.520, P = 0.506). Significant improvements in PEI-2 scores among the TFES group suggested good responsiveness (P < 0.001). The difference in change of PEI-2 scores between TFES and control was significant (P = 0.008), indicating good sensitivity. CONCLUSIONS: This study supports the validity, reliability, sensitivity and responsiveness of PEI-2 in measuring changes in enablement, making it a promising tool for evaluating enablement in cohort and intervention studies.

Control of Glucocorticoid Receptor Levels by PTEN Establishes a Failsafe Mechanism for Tumor Suppression.

The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (P-Rex1) promotes mammary tumor initiation and metastasis.

The Rac-GEF, P-Rex1, activates Rac1 signaling downstream of G protein-coupled receptors and PI3K. Increased P-Rex1 expression promotes melanoma progression; however, its role in breast cancer is complex, with differing reports of the effect of its expression on disease outcome. To address this we analyzed human databases, undertook gene array expression analysis, and generated unique murine models of P-Rex1 gain or loss of function. Analysis of PREX1 mRNA expression in breast cancer cDNA arrays and a METABRIC cohort revealed that higher PREX1 mRNA in ER+ve/luminal tumors was associated with poor outcome in luminal B cancers. Prex1 deletion in MMTV-neu or MMTV-PyMT mice reduced Rac1 activation in vivo and improved survival. High level MMTV-driven transgenic PREX1 expression resulted in apicobasal polarity defects and increased mammary epithelial cell proliferation associated with hyperplasia and development of de novo mammary tumors. MMTV-PREX1 expression in MMTV-neu mice increased tumor initiation and enhanced metastasis in vivo, but had no effect on primary tumor growth. Pharmacological inhibition of Rac1 or MEK1/2 reduced P-Rex1-driven tumoroid formation and cell invasion. Therefore, P-Rex1 can act as an oncogene and cooperate with HER2/neu to enhance breast cancer initiation and metastasis, despite having no effect on primary tumor growth.

Loss of Bcl-G, a Bcl-2 family member, augments the development of inflammation-associated colorectal cancer.

Gastrointestinal epithelial cells provide a selective barrier that segregates the host immune system from luminal microorganisms, thereby contributing directly to the regulation of homeostasis. We have shown that from early embryonic development Bcl-G, a Bcl-2 protein family member with unknown function, was highly expressed in gastrointestinal epithelial cells. While Bcl-G was dispensable for normal growth and development in mice, the loss of Bcl-G resulted in accelerated progression of colitis-associated cancer. A label-free quantitative proteomics approach revealed that Bcl-G may contribute to the stability of a mucin network, which when disrupted, is linked to colon tumorigenesis. Consistent with this, we observed a significant reduction in Bcl-G expression in human colorectal tumors. Our study identifies an unappreciated role for Bcl-G in colon cancer.

Colon organoid formation and cryptogenesis are stimulated by growth factors secreted from myofibroblasts.

Although small intestinal epithelial stem cells form crypts when using intestinal culture conditions, colon stem cells usually form colonospheres. Colon mesenchymal cell feeder layers can stimulate colon crypts to form organoids and produce crypts. We have investigated whether conditioned medium from colon mesenchymal cells can also stimulate colonosphere and organoid cryptogenesis. We prepared conditioned medium (CM) from WEHI-YH2 cells (mouse colon myofibroblasts); the CM stimulated both colonosphere formation and organoid cryptogenesis in vitro. The colon organoid-stimulating factors in WEHI-YH2 CM are inactivated by heating and trypsin digestion and proteins can be concentrated by ultrafiltration. Both the colonosphere- and organoid cryptogenesis- stimulatory effects of the CM are independent of canonical Wnt and Notch signaling. In contrast, bone morphogenetic protein 4 (BMP4) abolishes colonosphere formation and organoid cryptogenesis. The Transforming Growth Factor beta (TGFß) Type I receptor kinase inhibitor (A83-01) stimulates colonosphere formation, whereas the Epidermal Growth Factor receptor (EGFR) kinase inhibitor (AG1478) reduces the formation of colonospheres, but in the presence of EGF, a "just-right" concentration of AG1478 increases colon organoid cryptogenesis.

AT1 Receptor Modulator Attenuates the Hypercholesterolemia-Induced Impairment of the Myocardial Ischemic Post-Conditioning Benefits.

BACKGROUND AND OBJECTIVES: Ischemic post-conditioning (PostC) has been demonstrated as a novel strategy to harness nature's protection against myocardial ischemia-reperfusion (I/R). Hypercholesterolemia (HC) has been reported to block the effect of PostC on the heart. Angiotensin II type-1 (AT1) modulators have shown benefits in myocardial ischemia. The present study investigates the effect of a novel inhibitor of AT1, azilsartan in PostC of the heart of normocholesterolemic (NC) and HC rats. MATERIALS AND METHODS: HC was induced by the administration of high-fat diet to the animals for eight weeks. Isolated Langendorff's perfused NC and HC rat hearts were exposed to global ischemia for 30 min and reperfusion for 120 min. I/R-injury had been assessed by cardiac hemodynamic parameters, myocardial infarct size, release of tumor necrosis factor-alpha troponin I, lactate dehydrogenase, creatine kinase, nitrite in coronary effluent, thiobarbituric acid reactive species, a reduced form of glutathione, superoxide anion, and left ventricle collagen content in normal and HC rat hearts. RESULTS: Azilsartan post-treatment and six episodes of PostC (10 sec each) afforded cardioprotection against I/R-injury in normal rat hearts. PostC protection against I/R-injury was abolished in HC rat hearts. Azilsartan prevented the HC-mediated impairment of the beneficial effects of PostC in I/R-induced myocardial injury, which was inhibited by L-N5-(1-Iminoethyl)ornithinehydrochloride, a potent inhibitor of endothelial nitric oxide synthase (eNOS). CONCLUSION: Azilsartan treatment has attenuated the HC-induced impairment of beneficial effects of PostC in I/R-injury of rat hearts, by specifically modulating eNOS. Azilsartan may be explored further in I/R-myocardial injury, both in NC and HC conditions, with or without PostC.

Colonic myofibroblast cell line stimulates colonoid formation.

A stable and efficient system for the culture of murine colon epithelial cells or crypts is required to facilitate studies of the dynamics and factors affecting colon stem cell niche and crypt formation. Survival of colonic epithelial cells or crypts in vitro was not established until recently, when it was found that exogenous Wnt3A and R-spondin could promote cell survival and formation of spheroids (colonospheres) or some advanced organoids with well-developed crypts (colonoids). However, after 6-8 days in these culture conditions, only small numbers of colonospheres form organoids with crypt-like structures (colonoids). This study describes the use of a myofibroblast cell line and a coculture system that increases the efficiency of colonoid formation from isolated crypts. The enhanced coculture system has significantly improved colonoid-forming efficiency compared with results from previous systems. Crypt formation can be detected as early as day 2. The coculture system will facilitate the characterization of the colon stem cell niche and the changes that occur as a result of perturbations or mutations in colon stem or epithelial cells, such as those that favor precancerous adenoma or cancer.

Modulatory effects and action mechanisms of tryptanthrin on murine myeloid leukemia cells.

Leukemia is the disorder of hematopoietic cell development and is characterized by an uncoupling of cell proliferation and differentiation. There is a pressing need for the development of novel tactics for leukemia therapy as conventional treatments often have severe adverse side effects. Tryptanthrin (6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) is a naturally-occurring, weakly basic alkaloid isolated from the dried roots of medicinal indigo plants (Ban-Lan-Gen). It has been reported to have various biological and pharmacological activities, including anti-microbial, anti-inflammatory, immunomodulatory and anti-tumor effects. However, its modulatory effects and action mechanisms on myeloid cells remain poorly understood. In this study, tryptanthrin was shown to suppress the proliferation of the murine myeloid leukemia WEHI-3B JCS cells in a dose- and time-dependent manner. It also significantly reduced the growth of WEHI-3B JCS cells in vivo in syngeneic BALB/c mice. However, it exhibited no significant direct cytotoxicity on normal murine peritoneal macrophages. Flow cytometric analysis showed an obvious cell cycle arrest of the tryptanthrin-treated WEHI-3B JCS cells at the G0/G1 phase. The expression of cyclin D2, D3, Cdk 2, 4 and 6 genes in WEHI-3B JCS cells was found to be down-regulated at 24 h as measured by RT-PCR. Morphological and functional studies revealed that tryptanthrin could induce differentiation in WEHI-3B JCS cells, as shown by the increases in vacuolation, cellular granularity and NBT-reducing activity in tryptanthrin-treated cells. Collectively, our findings suggest that tryptanthrin might exert its anti-tumor effect on the murine myelomonocytic leukemia WEHI-3B JCS cells by causing cell cycle arrest and by triggering cell differentiation.

Effect of Cl- on tyrosinase: complex inhibition kinetics and biochemical implication.

Tyrosinase plays a core role in melanogenesis of the various organisms. Therefore, the regulation of the tyrosinase activity is directly related with melanin synthesis. In this study, we investigated the Cl(-)-induced inhibition of human tyrosinase and the potent role of Cl(-) as a negative regulator in melanogenesis. For the inhibition kinetic studies, human tyrosinase was differently prepared from the TXM13 melanotic cells as well as from cells that had undergone gene transfection. We found that Cl(-) inhibited tyrosinase in a slope-parabolic competitive manner and tyrosinase gene transfection into HEK293 cell significantly down-regulated the expression levels of solute carrier family 12, member 4 (potassium/chloride transporters, SLC12A7) and solute carrier family 12, member 7 (potassium/chloride transporters, SLC12A7), which are known to be Cl(-) transporters. From the results of the inhibition kinetic studies and the Cl(-) transporter expression level, we suggested that Cl(-) might act as a potent regulatory factor in melanogenesis. It is worth notice that a high content of Cl(-) exists physiologically and tyrosinase reacts sensitively to Cl- in a complex interaction manner.

Postural neck pain: an investigation of habitual sitting posture, perception of 'good' posture and cervicothoracic kinaesthesia.

Impairments of cervico-cephalic kinaesthesia and habitual forward head posture have been considered important in the aetiology of postural neck pain, yet these factors have not been specifically examined in a homogeneous clinical population. The objective of this study was to compare the habitual sitting posture (HSP), perception of good posture and postural repositioning error (PRE) of the cervico-thoracic (CT) spine in individuals with postural neck pain, with a matched group of asymptomatic subjects. Twenty-one subjects with postural neck pain and 22 asymptomatic control subjects were recruited into the study. An optical motion analysis system was used to measure the HSP and perceived 'good' sitting posture. PRE was measured over six trials where the subject attempted to replicate their self-selected 'good' posture. There was no difference between the groups in the HSP but significant differences were identified in the perception of 'good' posture. Posture repositioning error was higher for the head posture variables than for CT and shoulder girdle variables in both groups. However, there was no significant difference in posture repositioning error between groups for any of the posture measures. The findings suggest that individuals with postural neck pain may have a different perception of 'good' posture, but no significant difference in HSP or kinaesthetic sensibility compared with matched asymptomatic subjects.