Selective PI3K Inhibitor Research Articles

Selective inhibition of the PI3K isoform p110alpha using BYL719 protects against tyrosine kinase-mediated processes in PASMCs and reduces experimental pulmonary hypertension in mice and rats

Abstract Rationale Pulmonary arterial hypertension (PAH) is a vascular disease characterized by chronic increases in pulmonary vascular resistance (PVR), pulmonary arterial pressure (PAP), and right ventricular (RV) hypertrophy. Increased activation of receptor tyrosine kinase (RTK) -mediated signaling pathways leads to increased proliferation and migration of pulmonary smooth vascular muscle cells (PASMCs) which promote vascular remodeling processes. We identified the catalytic subunit p110alpha of phosphatidylinositol-3-kinase as a key enzyme for these processes and showed that both genetic ablation of p110alpha in SMCs and pharmacological inhibition can prevent experimental PH. Here, the effects of the orally bioavailable p110alpha selective PI3K inhibitor BYL719 on the RTK-mediated proliferation and chemotaxis of PASMCs, as well as the effects in the hypoxia-induced mouse and in the Sugen / hypoxia (SuHx) -induced rat model of PH were investigated. Methods Human and murine PASMCs were pretreated with different concentrations of BYL719 and stimulated with a mixture of growth factors (PDGF [30ng/ml], EGF [0,5ng/ml], bFGF [2ng/ml], insulin [0,5ng/ml], and FBS [5%]). Proliferation was investigated using a BrdU incorporation ELISA assay (Roche). Chemotaxis was quantified using modified Boyden chambers. Male BL/6 mice were subjected to hypoxia (10% O2) for 21 days. Treatment with BYL719 (or vehicle) was carried out via daily gavage of 50mg/kg bodyweight. In addition, a therapeutic approach was investigated using male Sprague Dawley rats in the SuHx model, which were treated with BYL719 (20 mg / kg body weight) or vehicle for 2 weeks after a three-week hypoxia phase. The RV pressure (RVSP) was measured using a Millar® or liquid-filled catheter. The RV hypertrophy is shown as the quotient of the weights of the RV to the LV + septum (RV / (LV + S)). Results Growth factor-induced proliferation and chemotaxis of the PASMCs were significantly and concentration-dependently inhibited by BYL719. The exposure to hypoxia led to an increase of the RVSP (24.5±0.95 to 35.2±1.28 mmHg) and the development of right ventricular hypertrophy (RV / LV + S 0.24±0.01 to 0.37±0.073), which was significantly reduced in the BYL719 treated group (RVSP 31.4±0.53 mmHg; RV / LV + S 0.31±0.01) (p<0.05). In addition, SuHx led to a robust increase of the RVSP (129.2±5.4 mmHg) and pronounced RV hypertrophy (RV / (LV + S): 0.86±0.04), which were significantly reduced by the therapeutic BYL719 treatment (102.0±6.1 mmHg or 0.64±0.03). Conclusion These results show that inhibition of p110alpha using the BYL719 reduced growth factor-mediated pathological processes in PASMCs in vitro, as well as hypoxia-induced (mouse) and already established SuHx-induced PH (rat). Thus, the inhibition of p110a using BYL719 represents a promising approach for the treatment of PAH. Funding Acknowledgement Type of funding sources: None.

Preclinical and phase I studies of KA2237, a selective and potent inhibitor of PI3K β/δ in relapsed refractory B cell lymphoma

PI3-kinase p110δ is mainly expressed in lymphocytes and is an attractive therapeutic target in B cell lymphomas. Targeting p110β may further suppress tumor growth and overcome escape mechanisms. KA2237 is an oral, potent, dual p110β/p110δ inhibitor. In preclinical studies, KA2237 inhibited p110β- and p110δ-dependent AKT activation and suppressed proliferation of diverse hematological and epithelial tumors. Twenty-one patients received KA2237 in a first-in-human phase I study (NCT02679196; diffuse large B cell, n = 8; follicular, n = 5; mantle cell, n = 3; chronic lymphocytic leukemia/small lymphocytic lymphoma, n = 3; marginal zone, n = 1; Waldenstrom’s, n = 1). Median age 69; median prior therapies 3. Eighty-six percent of patients experienced treatment-related adverse events (TRAEs). Forty-three percent of patients experienced grade ≥3 TRAEs, with rash (n = 3), pneumonia (n = 3), transaminitis (n = 2), and pneumonitis (n = 2) being most common. Thirty-three percent discontinued treatment due to adverse events. KA2237 induced objective responses in indolent and aggressive lymphoma (overall response rate 37%; complete response n = 4, partial response n = 3).

Abstract 291: Development of optimized chemical probes targeting PI3Ka to deconvolute the role of class I PI3Ks isoforms in insulin signaling

Abstract Phosphatidylinositol-3-kinase (PI3K) activity is aberrant in tumors, and PI3K inhibitors are investigated as cancer therapeutics.[1-5] The major obstacles to the successful implementation of PI3K-targeted cancer therapy are on-target adverse effects on insulin signaling. Isotype selective PI3K inhibitors have been exploited to answer fundamental questions regarding the role of PI3K isoforms in cell biology. However, the availability of claimed isoform-selective PI3Kα inhibitors is limited to BYL719 (Alpelisib)[6] and GDC0032 (Taselisib)[7], which do not maintain PI3Kα selectivity at a concentration required in cellular experimental settings and clinical applications. Highly selective PI3Kα inhibitors are expected to represent ideal tools to elucidate the role of PI3Kα isoform in tumor development and insulin signaling. As the systemic inactivation of PI3Kα is embryonic lethal, genetic approaches are currently limited to organ-specific targeting, and a specific inactivation of PI3Kα in an adult organism has not been achieved up-to-date. Herein, we generate high-quality PI3Kα chemical probes to dissect the role of PI3Kα in cancer and metabolism. We exploit covalent inhibitors, permanently blocking target functions, as a strategy to enhance the ligand binding selectivity for proteins in the same family. The non-conserved nucleophilic amino acid Cys862 in PI3Kα represents a promising target for covalent modifiers. We converted the reversible scaffold of PQR514[5] into irreversible compounds. An extensive Structure Activity Relationship (SAR) study was performed using CNX-1351[8] reacting group and introducing different heteroaliphatic rings in the linker. X-ray crystallography and bottom-up LC-MS/MS based proteomics validated the covalent modification of Cys862. Our pilot chemical probes exceeded in vitro and cellular potency over CNX-1351. The generation of a novel class of covalent PI3Kα-specific inhibitors with improved selectivity and persistency of PI3Kα-inhibition will shed light on the role of PI3Kα in cancer and metabolism. Our results will pave the way for the dissociation of PI3Ki antitumor activity from adverse effects on insulin action. [1] Beaufils F et al. J Med Chem. 2017, 60 (17), 7524-7538. [2] Rageot R et al. J Med Chem. 2019, 62 (13), 6241-6261. [3] Wymann MP et al. Nat Rev Mol Cell Biol. 2008, 9 (2), 62-176. [4] Marone R et al. Biochim Biophys Acta. 2008, 1784 (1), 159-185. [5] Borsari C et al. ACS Med Chem Lett. 2019, 10 (10), 1473-1479. [6] Markham A Drugs 2019, 79 (11), 1249-1253. [7] Zumsteg ZS et al. Clin Cancer Res. 2019, 22 (8), 2009-2019. [8] Nacht M et al. J Med Chem. 2013, 56 (3), 712-721. Citation Format: Martina De Pascale, Chiara Borsari, Erhan Keles, Jacob McPhail, Alexander Schäfer, Rohitha Sriramaratnam, Matthias Gstaiger, John Burke, Matthias Wymann. Development of optimized chemical probes targeting PI3Ka to deconvolute the role of class I PI3Ks isoforms in insulin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 291.

Notoginsenoside R1 Improves Cerebral Ischemia/Reperfusion Injury by Promoting Neurogenesis via the BDNF/Akt/CREB Pathway.

Notoginsenoside R1 (R1), a major component isolated from P. notoginseng, is a phytoestrogen that exerts many neuroprotective effects in a rat model of ischemic stroke. However, its long-term effects on neurogenesis and neurological restoration after ischemic stroke have not been investigated. The aim of this study was to evaluate the effects of R1 on neurogenesis and long-term functional recovery after ischemic stroke. We used male Sprague-Dawley rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). R1 was administered by intraperitoneal (i.p.) injection immediately postischemia. We showed that R1 significantly decreased infarct volume and neuronal loss, restored neurological function, and stimulated neurogenesis and oligodendrogenesis in rats subjected to MCAO/R. More importantly, R1 promoted neuronal proliferation in PC12 cells in vitro. The proneurogenic effects of R1 were associated with the activation of Akt/cAMP responsive element-binding protein, as shown by the R1-induced increase in brain-derived neurotrophic factor (BDNF) expression, and with the activation of neurological function, which was partially eliminated by selective inhibitors of BDNF and PI3K. We demonstrated that R1 is a promising compound that exerts neuroprotective and proneurogenic effects, possibly via the activation of BDNF/Akt/CREB signaling. These findings offer insight into exploring new mechanisms in long-term functional recovery after R1 treatment of ischemic stroke.

Sevoflurane alleviates oxygen-glucose deprivation/reoxygenation-induced injury in HT22 cells through regulation of the PI3K/AKT/GSK3β signaling pathway.

Sevoflurane (Sev), a volatile anesthetic, has been reported to exhibit beneficial effects on different ischemia/reperfusion (I/R)-injured organs. However, the neuroprotective effect of Sev on cerebral I/R injury is poorly understood. In the present study, the effects of Sev on HT22 cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R) injury are investigated. The present study demonstrated that OGD/R suppressed the cell viability and increased lactate dehydrogenase (LDH) release from the cells, and these effects were attenuated by Sev treatment. The results also demonstrated that Sev alleviated OGD/R-induced cell apoptosis via flow cytometry and caspase-3 activity determination. Biochemical analysis results revealed that Sev significantly protected against OGD/R-induced oxidative stress by reducing ROS generation and improving antioxidant defense markers. Western blot analysis demonstrated that Sev reactivated the PI3K/AKT/glycogen synthase kinase-3β (GSK3β) signaling pathway, which was inhibited by OGD/R. In addition, wortmannin, a selective PI3K inhibitor was used to investigate the underlying pathways. Notably, the neuroprotective effect of Sev on apoptosis and reactive oxygen species production was found to be suppressed by wortmannin. Collectively, these results demonstrated that Sev may protect neuronal cells against OGD/R-induced injury through the activation of the PI3K/AKT/GSK3β signaling pathway. The findings from the present study provide a novel insight into understanding the neuroprotective effect of Sev on cerebral I/R injury.

Research update on the anticancer effects of buparlisib.

Buparlisib is a highly efficient and selective PI3K inhibitor and a member of the 2,6-dimorpholinopyrimidine-derived family of compounds. It selectively inhibits four isomers of PI3K, PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ, by competitively binding the lipid kinase domain on adenosine 5'-triphosphate (ATP), and serves an important role in inhibiting proliferation, promoting apoptosis and blocking angiogenesis, predominantly by antagonizing the PI3K/AKT pathway. Buparlisib has been confirmed to have a clinical effect in patients with solid tumors and hematological malignancies. A global, phase II clinical trial with buparlisib and paclitaxel in head and neck squamous cell carcinoma has now been completed, with a manageable safety profile. Buparlisib currently has fast-track status with the United States Food and Drug Administration. The present review examined the biochemical structure, pharmacokinetic characteristics, preclinical data and ongoing clinical studies of buparlisib. The various mechanisms of influence of buparlisib in tumors, particularly in preclinical research, were summarized, providing a theoretical basis and direction for basic research on and clinical treatment with buparlisib.

Triplet Therapy with Palbociclib, Taselisib, and Fulvestrant inPIK3CA-Mutant Breast Cancer and Doublet Palbociclib and Taselisib in Pathway-Mutant Solid Cancers

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1.

A novel peptide relieves endothelial cell dysfunction inpreeclampsia by regulating the PI3K/mTOR/HIF1α pathway.

Preeclampsia (PE) is a pregnancy-specific complication characterized by hypertension and proteinuria, and it is one of the primary global causes of maternal and perinatal mortality. Poor remodeling of placental arteries and endothelial dysfunction serve important roles in the pathogenesis of PE. Peptide derived from complement C4 A chain (PDCC4) was identified in our previous peptidome analysis of serum from patients with PE. The present study aimed to investigate the effect of PDCC4 on endothelial dysfunction in PE. TNF-α stimulated HUVECs were employed to mimic endothelial dysfunction in PE, and Cell Counting Kit 8 assay, wound healing assay, tube formation assay, RNA-sequencing (seq) and western blot analysis were performed using HUVECs. Moreover, an in vivo model of PE was established using pregnant rats treated with lipopolysaccharide (LPS), and blood pressure monitoring, histopathological examination, ELISA and immunohistochemistry were performed on rats. It was found that TNF-α impaired proliferation, migration and tube formation of HUVECs, but pretreatment with PDCC4 moderated these effects. RNA-seq and western blotting demonstrated that the PI3K/mTOR/HIF1α signaling pathway was activated by PDCC4, and a selective PI3K inhibitor reversed the protective function of PDCC4 on TNF-α stimulated HUVECs. Additionally, PDCC4 alleviated hypertension, histopathological changes of placenta and kidney and the expression levels of endothelial injury markers and inflammatory cytokines induced by LPS in rats. These results suggested that PDCC4 relieved endothelial dysfunction in PE via PI3K/mTOR/HIF1α signaling pathway and may be a potential therapy for PE.

Phase III randomized study of taselisib or placebo with fulvestrant in estrogen receptor-positive, PIK3CA-mutant, HER2-negative, advanced breast cancer: the SANDPIPER trial

The phase III SANDPIPER study assessed taselisib (GDC-0032), a potent, selective PI3K inhibitor, plus fulvestrant in estrogen receptor-positive, HER2-negative, PIK3CA-mutant locally advanced or metastatic breast cancer. Postmenopausal women with disease recurrence/progression during/after an aromatase inhibitor were randomized 2 : 1 to receive taselisib (4 mg; taselisib arm) or placebo (placebo arm) plus fulvestrant (500 mg). Stratification factors were visceral disease, endocrine sensitivity, and geographic region. Patients with PIK3CA-mutant tumors (central cobas® PIK3CA Mutation Test) were randomized separately from those without detectable mutations. The primary endpoint was investigator-assessed progression-free survival (INV-PFS) in patients with PIK3CA-mutant tumors. Secondary endpoints included objective response rate, overall survival, clinical benefit rate, duration of objective response, PFS by blinded independent central review (BICR-PFS), safety, and time to deterioration in health-related quality of life. The PIK3CA-mutant intention-to-treat population comprised 516 patients (placebo arm: n= 176; taselisib arm: n= 340). INV-PFS was significantly improved in the taselisib {7.4 months [95% confidence interval (CI), 7.26-9.07]} versus placebo arm (5.4 months [95% CI, 3.68-7.29]) (stratified hazard ratio [HR] 0.70; 95% CI, 0.56-0.89; P= 0.0037) and confirmed by BICR-PFS (HR 0.66). Secondary endpoints, including objective response rate, clinical benefit rate, and duration of objective response, showed consistent improvements in the taselisib arm. Safety was assessed in all randomized patients who received at least one dose of taselisib/placebo or fulvestrant regardless of PIK3CA-mutation status (n= 629). Serious adverse events were lower in the placebo versus taselisib arm (8.9% versus 32.0%). There were more discontinuations (placebo arm: 2.3%; taselisib arm: 16.8%) and dose reductions (placebo arm: 2.3%; taselisib arm: 36.5%) in the taselisib arm. SANDPIPER met its primary endpoint; however, the combination of taselisib plus fulvestrant has no clinical utility given its safety profile and modest clinical benefit.

Identification of methyl (5-(6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-4-morpholinopyrrolo[2,1-f][1,2,4]triazin-2-yl)-4-(trifluoromethyl)pyridin-2-yl)carbamate (CYH33) as an orally bioavailable, highly potent, PI3K alpha inhibitor for the treatment of advanced solid tumors.

In various human cancers, PI3Ks pathway is ubiquitously dysregulated and thus become a promising anti-cancer target. To discover new potent and selective PI3K inhibitors as potential anticancer drugs, new pyrrolo[2,1-f][1,2,4]triazines were designed, leading to the discovery of compound 37 (CYH33), a selective PI3Kα inhibitor (IC50=5.9nM, β/α, δ/α,γ/α=101-, 13-, 38-fold). Western blot analysis confirmed that compound 37 could inhibit phosphorylation of AKT in human cancer cells to modulate the cellular PI3K/AKT/mTOR pathway. And further evaluation invivo against SKOV-3 xenograft models demonstrated that a dose-dependent antitumor efficacy was achieved.

266 Poster - Impact of BMI on the outcome of metastatic breast cancer patients treated with everolimus: A retrospective exploratory analysis of the BALLET study

Introduction Reliable biomarkers of response to mTOR inhibition are yet to be identified. As mTOR is heavily implicated in cell-metabolism, we investigated the relation between BMI variation and outcomes in metastatic breast cancer (mBC) patients treated with everolimus. Results we found a linear correlation between everolimus exposure duration and BMI/weight decrease. Patients exhibiting >2 kg weight loss or >3% BMI decrease from baseline at the end of treatment (EOT) had a statistically significant improvement in PFS. Interestingly, a similar BMI/weight decrease within the first 8 weeks of therapy identified patients at higher risk of progression. Patients and methods we performed a retrospective analysis of patients enrolled in the BALLET trial who progressed during the study. Primary end-point was progression-free survival (PFS). Secondary end-point was the identification of other predictors of response. Conclusion A >3% weight loss at EOT is associated with better outcome in mBC patients treated with everolimus. On the contrary, a significant early weight loss represents a predictor of poor survival and could therefore be used as an early negative prognostic marker. As PI3K-inhibition also converges onto mTOR, these findings might extend to patients treated with selective PI3K inhibitors and warrant further investigation.

Frailty Is Associated With Neutrophil Dysfunction Which Is Correctable With Phosphoinositol-3-Kinase Inhibitors.

Neutrophil dysfunction has been described with age, appears exaggerated in infection, with altered phosphoinositol signaling a potential mechanism. However, functional aging is heterogeneous. Frailty is a negative health status and is more common in older adults. We hypothesized that neutrophil migration may be compromised in frailty, associated with the degree of frailty experienced by the older person. We compared measures of frailty, neutrophil function, and systemic inflammation in 40 young and 77 older community-dwelling adults in the United Kingdom. Systemic neutrophils exhibited an age-associated reduction in the accuracy of migration (chemotaxis) which was further blunted with frailty. The degree of migratory inaccuracy correlated with physical (adjusted hand grip strength) and cognitive (Stroop test) markers of frailty. Regression analysis demonstrated that age, Charlson comorbidity index, and frailty index were able to predict neutrophil chemotaxis. Reduced chemotaxis of neutrophils from frail adults could be reversed using selective PI3K inhibitors. Exposure of neutrophils from young adults to plasma from chronically inflamed frail older adults could not recapitulate the migratory deficit in vitro, and there were no relationships with systemic inflammation and neutrophil dysfunction. Frailty exaggerated the neutrophil deficits seen with advanced age but aspects of the frailty-associated deficit in neutrophil function are rescuable and thus potentially form a therapeutic target to improve outcomes from infection in older adults.

Impact of BMI on the outcome of metastatic breast cancer patients treated with everolimus: a retrospective exploratory analysis of the BALLET study.

Introduction: Reliable biomarkers of response to mTOR inhibition are yet to be identified. As mTOR is heavily implicated in cell-metabolism, we investigated the relation between BMI variation and outcomes in metastatic breast cancer (mBC) patients treated with everolimus.Results: we found a linear correlation between everolimus exposure duration and BMI/weight decrease. Patients exhibiting >2 kg weight loss or >3% BMI decrease from baseline at the end of treatment (EOT) had a statistically significant improvement in PFS. Interestingly, a similar BMI/weight decrease within the first 8 weeks of therapy identified patients at higher risk of progression.Patients and methods: we performed a retrospective analysis of patients enrolled in the BALLET trial who progressed during the study. Primary end-point was progression-free survival (PFS). Secondary end-point was the identification of other predictors of response.Conclusion: A >3% weight loss at EOT is associated with better outcome in mBC patients treated with everolimus. On the contrary, a significant early weight loss represents a predictor of poor survival and could therefore be used as an early negative prognostic marker. As PI3K-inhibition also converges onto mTOR, these findings might extend to patients treated with selective PI3K inhibitors and warrant further investigation

Troxerutin attenuates oxygen‑glucose deprivation and reoxygenation‑induced oxidative stress and inflammation by enhancing the PI3K/AKT/HIF‑1α signaling pathway in H9C2 cardiomyocytes.

Myocardial ischemia-reperfusion (MI/R) injury is a complex pathological process that occurs when tissues are reperfused following a prolonged period of ischemia. Troxerutin has been reported to have cardioprotective functions. However, the underlying mechanism by which troxerutin protects against MI/R injury has not been fully elucidated. The aim of the present study was to explore whether troxerutin-mediated protection against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced H9C2 cell injury was associated with the inhibition of oxidative stress and the inflammatory response by regulating the PI3K/AKT/hypoxia-inducible factor-1α (HIF-1α) signaling pathway. The results of the present study suggested that troxerutin pretreatment prevented the OGD/R-induced reduction in cell viability, and the increase in lactate dehydrogenase activity and apoptosis. Troxerutin reversed OGD/R-induced the inhibition of the PI3K/AKT/HIF-1α signaling pathway as demonstrated by the increased expression of PI3K and HIF-1α, and the increased ratio of phosphorylated AKT/AKT. LY294002, a selective PI3K inhibitor, inhibited the PI3K/AKT/HIF-1α signaling pathway and further attenuated the protective effect of troxerutin against OGD/R-induced H9C2 cell damage. Furthermore, small interfering (si)RNA-mediated knockdown of HIF-1α reduced troxerutin-induced protection against OGD/R injury. Troxerutin pretreatment alleviated OGD/R-induced oxidative stress, as demonstrated by the reduced generation of reactive oxygen species and malonaldehyde content, and the increased activities of superoxide dismutase and glutathione peroxidase, which were reduced by HIF-1α-siRNA. Troxerutin-induced decreases in the levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α in OGD/R conditions were also reduced by HIF-1α-siRNA. The results from the present study indicated that troxerutin aggravated OGD/R-induced H9C2 cell injury by inhibiting oxidative stress and the inflammatory response. The primary underlying protective mechanism of troxerutin was mediated by the activation of the PI3K/AKT/HIF-1α signaling pathway.

Preclinical evaluation of LX-086, a small molecule as a selective inhibitor of PI3K.

e15662 Background: PI3K/Akt signaling pathway plays a key role in a series of cellular functions related to cell growth, proliferation, survival and differentiation. PI3Kα is ubiquitously expressed, and associates with angiogenesis and glucose homeostasis. Gain of function mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are among the most common somatic alterations in solid tumors. The purpose of this study is to investigate the in vitro/ in vivo antitumor activity, and safety of LX-086, a small molecule of PI3Kα selective inhibitor, in preclinical models of solid cancer with PIK3CA mutation or amplification. Methods: Kinase inhibitory activity and selectivity of LX-086 were determined with PI3K kinase assays. Cellular pAktS473 inhibitory activity and selectivity were evaluated in BT-474, MDA-MB-468, RAW264.7 and Jeko-1 cell lines (cell lines PI3Kα/β/γ/δ are specifically highly expressed respectively). The antitumor activity of LX-086 was evaluated in vivo in PIK3CA amplified or mutant breast cancer BT-474, T47D and ovarian cancer SK-OV-3 cell-derived mouse xenograft (CDX) models. Results: LX-086 displayed potent kinase inhibitory activity for WT PI3Kα, PI3KαE545K, PI3KαH1047R with IC50 1.80 nM/1.13 nM/0.69 nM, respectively. LX-086 inhibited cell pAktS473 in BT-474 cells with IC50 35.4 nM. The selectivity of LX-086 in PI3Kα over PI3Kβ/δ/γ is similar with that of alpelisib, which had been approved by FDA in 2019. LX-086 showed antitumor efficacy in HER2+/HR+, PIK3CA amplified breast cancer BT-474 CDX model (TGI = 106% @40 mpk, QD), as well as in the HER2-/HR+, PIK3CA H1047R breast cancer T47D CDX model ( TGI = 84.7%@ 40 mpk, QD) and PIK3CA H1047R ovarian cancer SK-OV-3 CDX model ( TGI = 69.2%@ 40 mpk, QD). The 28-days toxicity study in dog also indicated that LX-086 had similar therapeutic index with alpelisib, with low risk of hyperglycemia. Conclusions: We have identified a novel potent and safe PI3Kα inhibitor LX-086. Preclinical studies show antitumor efficacy of LX-086 in PIK3CA mutant and amplified solid cancer models. LX-086 represents a promising clinical candidate for the treatment of solid cancers with PIK3CA mutation or amplification.

Biallelic loss of FAM46C triggers tumor growth with concomitant activation of Akt signaling in multiple myeloma cells

Loss of heterozygosity or mutation of the family with sequence similarity 46, member C (FAM46C) gene on chromosome band 1p12 is associated with shorter overall survival of patients with multiple myeloma (MM). In this study, using human MM cell lines (KMS‐11, OCI‐My5, and ANBL‐6), we generated FAM46C−/− cell clones and examined the effect of disruption of FAM46C on cell survival and cellular signaling. Cell proliferation assays showed increased clonogenicity of FAM46C−/− KMS‐11 cells compared to WT cells. Xenograft experiments showed significantly shorter overall survival of mice harboring the FAM46C−/− cell‐derived tumors than mice with the FAM46CWT cell‐derived tumors. Notably, levels of phosphorylated Akt and its substrates increased both in vitro and in vivo in the FAM46C−/− cells compared to WT cells. In addition, caspase activities decreased in the FAM46C−/− cells. Results of gene set enrichment analysis showed that loss of FAM46C significantly activated serum‐responsive genes while inactivating phosphatase and tensin homolog (PTEN)‐related genes. Mechanistically, loss of FAM46C decreased the PTEN activity, number of apoptotic cells, and caspase activities. PF‐04691502, a selective PI3K inhibitor, suppressed the augmented phosphorylation of Akt and its substrate FoxO3a. Treatment with afuresertib (a specific Akt inhibitor) in combination with bortezomib additively decreased FAM46C−/− MM cell survival. Collectively, this study is the first to report that loss of FAM46C triggers the concomitant activation of the PI3K‐Akt signaling pathway, which might be a therapeutic target for MM with abnormalities in the FAM46C gene.

TREM2 Regulates Heat Acclimation-Induced Microglial M2 Polarization Involving the PI3K-Akt Pathway Following EMF Exposure.

The function of triggering receptor expressed on myeloid cells-2 (TREM2) has been described within microglia with a beneficial activated phenotype. However, the role of TREM2 underlying microglial phenotypic alterations in the cross-tolerance protection of heat acclimation (HA) against the inflammatory stimuli electromagnetic field (EMF) exposure is less well known. Here, we investigated the TREM2-related signaling mechanism induced by HA in EMF-stimulated N9 microglial cells (N9 cells). We found that EMF exposure significantly increased the production of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α, IL-1β, and IL-6), and the expression of M1 markers (CD11b and CD86); meanwhile, decreased the levels of anti-inflammatory cytokines (IL-4 and IL-10) and the expression of M2 markers (CD206 and Arg1) in N9 cells. Clearly, HA treatment decreased the secretion of TNF-α, IL-1β and IL-6 and the expression of CD11b and CD86, and enhanced the production of IL-4 and IL-10 and the expression of CD206 and Arg1. Moreover, TREM2 esiRNA and selective inhibitor of PI3K clearly decreased anti-inflammatory cytokines production, M2 markers expression, and phosphorylation of PI3K and Akt following HA plus EMF stimulation. These results indicate that TREM2 and PI3K-Akt pathway are involved in the cross-tolerance protective effect of HA in microglial polarization towards the EMF exposure. This finding inspires future studies that aim to explore the non-drug approaches underlying EMF stimulation and other central nervous system (CNS) inflammatory diseases.

IRS1/PI3K/AKT pathway signal involved in the regulation of glycolipid metabolic abnormalities by Mulberry (Morus alba L.) leaf extracts in 3T3-L1 adipocytes

BackgroundMulberry (Morus alba L.) leaf tea benefits the control of diabetes in Asian nations. This study was aim to investigate if the flavonoids, which extracts from mulberry leaves, could regulate the metabolism of glycolipid, and to investigate if flavonoids could regulate IRS1/PI3K/AKT pathway signal to affect the expression of FAS and membrane transfer capacity GLUT4 in 3T3-L1 adipocytes.ResultsResults revealed that flavonoids decreased the levels of free fatty acid and increased the glucose consumption and the levels of adiponectin and leptin in a dose-dependent manner, and remarkably increased the protein expression levels of p-IRS1, p-PI3K, p-Akt, total GLUT4, and membrane GLUT4, and decreased the protein expression levels of PTEN and FAS in 3T3-L1 adipocytes IR model. On the other hand, wortmannin (2 nM), a selective and irreversible PI3K inhibitor, significantly decreased the glucose consumption and the adiponectin and leptin levels, and increased the free fatty acid level in flavonoids treated 3T3-L1 adipocytes IR model. Furthermore, wortmannin (2 nM) partly eliminated the activation of PI3K/AKT signaling, the suppression of FAS, and the up-regulated membrane transfer capacity of GLUT4 in flavonoids treated 3T3-L1 adipocytes IR model.ConclusionIn conclusion, our results illustrated that mulberry leaf extracts flavonoids alleviated the glycolipid metabolic abnormalities in 3T3-L1 adipocytes IR model, and the effect was associated with the activation of IRS1/PI3K/AKT pathway, the suppression of FAS, and the up-regulation of membrane transfer capacity of GLUT4.

MST4 inhibits human hepatocellular carcinoma cell proliferation and induces cell cycle arrest via suppression of PI3K/AKT pathway.

Objective: MST4 has exhibited functions in regulating cell polarity, Golgi apparatus, cell migration, and cancer. Mechanistically, it affects the activity of p-ERK, Hippo-YAP pathway and autophagy. The aim of this study is to further examine the functions of MST4 in hepatocellular carcinoma (HCC) and the underlying mechanism.Methods: The expression level of MST4 in HCC and noncancer adjacent liver tissues was determined by qRT-PCR and immunohistochemistry staining. Wild-type MST4 (MST4) and a dominant-negative mutant of MST4 (dnMST4) were overexpressed in HCC cell lines, respectively. CCK-8 assay, EdU incorporation assay, and soft agar assay were used to determine cell proliferation in vitro. The xenograft mouse model was employed to determine HCC cell growth in vivo. Cell cycle analysis was performed by PI staining and flow cytometry. The expression of key members in PI3K/AKT pathway was detected by Western blot analysis.Results: In our study, we reported new evidence that MST4 was frequently down-regulated in HCC tissues. Gain-of-function and loss-of-function experiments demonstrated that MST4 negatively regulated in vitro HCC cell proliferation. Additionally, MST4 overexpression suppressed Bel-7404 cell tumor growth in nude mice. Further experiments revealed that the growth-inhibitory effect of MST4 overexpression was partly due to a G1-phase cell cycle arrest. Importantly, mechanistic investigations suggested that dnMST4 significantly elevated the phosphorylation levels of key members of PI3K/AKT pathway, and the selective PI3K inhibitor LY294002 can reverse the proliferation-promoting effect of dnMST4.Conclusions: Overall, our results provide a new insight into the clinical significance, functions and molecular mechanism of MST4 in HCC, suggesting that MST4 might have a potential therapeutic value in the HCC clinical treatment.

Anti-proliferative effects of a small molecule inhibitor of CDK AT7519 on chronic myeloid leukemia (CML) cells through halting the transition of cells from G2/M phase of the cell cycle

Pathogenesis of chronic myeloid leukemia (CML) has mostly been studied with regard to the oncogenic role of BCR/ABL fusion; however, recent disclosures have declared that the challenges with the treatment of CML patients would not be resolved until the role of other aberrancies is ignored. Given the involvement of cyclin-dependent kinases (CDKs) in the pathogenesis of CML, the present study aimed to investigate the effects of a multi-CDK inhibitor AT7519 on BCR/ABL-harboring CML-derived K562 cells. Our results showed that AT7519 effectively reduced the survival of K562 and induced its anti-proliferative effect through the induction of G2/M arrest due to elevated p21 and p27. The resulting data also revealed that either direct or indirect suppression of c-Myc using specific c-Myc inhibitor 10058-F4 and selective PI3K inhibitor CAL-101 resulted in a superior cytotoxicity, suggesting that the activation of PI3K pathway could attenuate antileukemic effects of the inhibitor, at least partly, through a c-Mycdependent mechanism. To the best of our knowledge, to date, no study has addressed the effect of autophagy on CML cell response to AT7519, and, herein, we proposed for the first time that the suppression of autophagy boosted AT7519 cytotoxicity against K562. Overall, we suggested that selective CDK inhibitor AT7519 exerted antileukemic effect against CML cells and propose a novel therapeutic application for the inhibitor either as a single agent or in combination with c-Myc and/or PI3K inhibitors.