Activation Of Phosphatidylinositol 3-kinase Pathway Research Articles

Expression of phosphorylated ribosomal protein S6 in mesothelioma patients - correlation with clinico-pathological characteristics and outcome: results from the European Thoracic Oncology Platform (ETOP) Mesoscape project

Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semi-quantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naïve and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis non-epithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p < 0.001, 10.7 versus 16.9 months) and PFS (p < 0.001, 6.2 versus 10.8 months). In subgroup analysis, in non-epithelioid PM patients high pS6 expression was associated with significantly shorter OS and PFS. These exploratory findings suggest a clinically relevant PI3K pathway activation in non-epithelioid PM which might lay the foundation for future targeted treatment strategies.

Long-term treatment of cancer-prone germline PTEN mutant micewith low-dose rapamycin extends lifespan and delays tumour development.

PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underlie PTEN hamartoma tumour syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralise the impact of PTEN loss, rendering this a more druggable context compared with those of other tumour suppressor pathways such as loss of TP53. However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short-term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6 weeks onwards to mice with heterozygous germline Pten loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay in, but not full blockade of, the development of a range of proliferative lesions, including gastro-intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long-term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract 1955: Simultaneous longitudinal assessment of PIK3CA genomic mutations and PI3K pathway activity in circulating tumor cells in metastatic breast cancer

Abstract Background: Phosphatidylinositol-3-kinase (PI3K) activating mutations are found in 30-40% of breast cancers, and the PI3K inhibitor alpelisib is FDA-approved for PIK3CA-mutated hormone-receptor positive metastatic breast cancer (MBC). However, rates of intrinsic alpelisib resistance are as high as 40%, and there is an ongoing need for novel biomarkers to help guide patient selection. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) may better reflect the heterogeneity of metastatic disease than tissue biopsy, and are amenable to longitudinal analysis. By combining targeted ctDNA sequencing with CTC transcriptional profiling and quantitation of PI3K pathway phospho-proteins, we have developed the first multiplex assay to simultaneously assess genomic alterations and CTC PI3K pathway activity via liquid biopsy. Methods: Peripheral blood was collected from MBC patients with known PIK3CA mutation status serially prior to starting new standard therapies and on treatment. Cell-free DNA was extracted from plasma and sequenced using a custom capture panel (IDT). CTCs were isolated with VERSA microfluidic technology integrating capture with downstream analysis. CTCs were captured immunomagnetically followed by RNA isolation on chip and RNA-seq, or staining on chip for phospho-AKT pS473 and total AKT or phospho-rpS6 pS235/S236 and total rpS6, followed by quantification of single cell phospho/total protein mean fluorescence ratios. Results: In a pilot cohort of 14 patients, mean CTC phospho-AKT/total AKT protein expression ratio was higher in patients with PIK3CA mutations compared to patients without mutations (0.37 vs 0.26, p&amp;lt;0.01). A transcriptional signature of PI3K activity was also higher in CTCs from patients with PIK3CA mutations than from patients without. Among patients with PIK3CA mutations, there was inter-patient heterogeneity in CTC phospho-AKT and phospho-rpS6, consistent with variability in pathway activation. One patient with a tissue PIK3CA mutation 5 years prior did not have the mutation detected in ctDNA at time of liquid biopsy, and had low phospho-AKT and transcriptional PI3K activity in CTCs. In serial sampling of a patient receiving a PI3K inhibitor, CTC phospho-AKT was decreased on treatment, and increased at time of progression. Conclusion: We have demonstrated the feasibility of a comprehensive liquid biopsy for simultaneous monitoring of PI3K pathway mutations in ctDNA and PI3K pathway signaling in CTCs via transcriptional profiling and phospho-protein expression in patients with MBC. Future work will prospectively evaluate this assay in patients receiving alpelisib for PIK3CA-mutated MBC. This has the potential to complement PIK3CA mutation status as a biomarker of sensitivity to PI3K therapies, and may also provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs on treatment. Citation Format: Marina N. Sharifi, Kyle T. Helzer, Jamie M. Sperger, Matthew L. Bootsma, Hannah Krause, Cole S. Gilsdorf, Serena K. Wolfe, Zachary Kauffman, Amye J. Tevaarwerk, Mark E. Burkard, Amanda M. Parkes, Ruth M. O'Regan, Kari B. Wisinski, Shuang G. Zhao, Joshua M. Lang. Simultaneous longitudinal assessment of PIK3CA genomic mutations and PI3K pathway activity in circulating tumor cells in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1955.

High incidence of PI3K pathway gene mutations in South Indian cervical cancers

IntroductionCervical cancer is the second most common cancer in India. The phosphatidylinositol-3 kinase (PI3K) signaling is one of the most commonly activated pathways in cancer and comprises key molecules commonly targeted in cancer therapy. This study analyzed six PI3K pathway gene mutations. MethodsWe carried out targeted next-generation sequencing of six PI3K pathway genes (PIK3CA, PIK3R1, PTEN, AKT1, TSC2, and mTOR) in a total of 93 South Indian cervical cancer samples and confirmed them by sanger sequencing. ResultsThe PI3K pathway gene mutations were observed in 54.8% (51/93) of the tumors and PIK3CA was the most mutated (34.4%, 32/93), followed by TSC2 (18.3%, 17/93), and PIK3R1 (14%, 13/93). The PIK3CA hotspot mutations E542K and E545K observed in this study were likely to disrupt the p110α-p85α interaction that could result in the PI3K pathway activation. We also found a few novel mutations in PIK3R1, PTEN, AKT1, TSC2, and mTOR genes while some of the tumors harbored multiple mutations in the genes of the PI3K pathway. The majority of the tumors were positive for high-risk HPV16/18 (60.7%). ConclusionsThe high incidence of the PI3K pathway gene mutations observed in this study could be exploited for the therapeutic management of cervical cancers.

Schizophrenia predisposition gene Unc-51-like kinase 4 for the improvement of cerebral ischemia/reperfusion injury.

Cerebral ischemia/reperfusion injury (CIRI) has complex pathogenesis, and inhibiting apoptosis and supporting neural progenitor proliferation are extremely beneficial strategies for treating CIRI. Unc-51-like kinase 4 (ULK4), a susceptibility gene for schizophrenia, promotes neural progenitors proliferation. The phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in CIRI via inhibition of apoptosis. Therefore, the relationship among ULK4, the PI3K pathway, and apoptosis in the context of CIRI has attracted our great interest. Primary cortical neurons were subjected to oxygen-glucose deprivation/reperfusion (OGD/R), and rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). Transfection of the ULK4-overexpression lentivirus was performed alone or in combination with PI3K inhibitor treatment. Here, we revealed that ULK4 was poorly expressed in the cortex in MCAO/R rats and OGD/R-treated primary cortical neurons, ULK4 overexpression inhibited apoptosis, and reduced neurological deficit scores, cerebral infarct volume, and histopathological damage. Moreover, ULK4 overexpression increased PI3K expression and the p-protein kinase B/AKT and p-glycogen synthase kinase 3 beta (GSK3β)/GSK3β ratios, and inhibited apoptosis, while a PI3K inhibitor reversed the effects of ULK4 overexpression on CIRI. ULK4 protects against CIRI, and the underlying mechanism involves PI3K pathway activation which in turn inhibits apoptosis.

B-cell antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

Mutations in the transcription factor FOXO1 mimic positive selection signals to promote germinal center B cell expansion and lymphomagenesis

The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.

Abstract PS5-35: Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies

Abstract Background: Somatic genomic alterations that activate the phosphatidylinositol-3-kinase (PI3K) pathway signaling are found in 30-50% of breast cancers, and the p110a-specific inhibitor alpelisib is approved for use in combination with fulvestrant in PIK3CA mutated hormone receptor-positive (HR+) metastatic breast cancer. However, preclinical and clinical data has demonstrated significant variability in response to alpelisib and other PI3K inhibitors even in the presence of hotspot PIK3CA activating mutations, likely reflecting functional differences between somatic alterations, bypass signaling mechanisms, and intratumor clonal heterogeneity. Thus, there is an ongoing need for novel predictive biomarkers to help guide patient selection for these therapies. Circulating tumor cells (CTCs) represent an accessible source of tumor derived analytes that allow for the interrogation of protein level readouts of PI3K pathway activation, may better reflect the biologic heterogeneity of metastatic disease than single site solid tumor biopsy, and are amenable to longitudinal analysis. Here, we report the development of an assay to evaluate the expression of activated AKT (AKT pS473) in CTCs as a putative biomarker of sensitivity to PI3K inhibitor therapies in metastatic breast cancer. Methods: Peripheral blood for CTC isolation was collected serially from patients with metastatic breast cancer and known somatic PI3K pathway mutation status. Samples were processed using VERSA (Versatile Exclusion-based Rare Sample Analysis), a microfluidic technology that integrates CTC capture and downstream analysis. Following fixation, CTCs were captured immunomagnetically with antibodies for EpCAM and Trop2, permeabilized and stained on chip for pan-AKT plus AKT pS473. Quantitative fluorescent microscopy was used to enumerate CTCs, defined as cytokeratin positive cells with intact nuclei and negative for a group of normal blood cell markers (CD45/CD11b/CD34/CD66b), and to quantify activated AKT protein expression in CTCs and matched peripheral blood mononuclear cells (PBMCs). Results: 100% of patients with somatic PIK3CA mutations in our pilot cohort had CTCs with detectable phospho-AKT expression, and in 50% of patients, the median phospho-AKT/pan AKT ratio was higher in CTCs compared to matched peripheral blood cells, suggestive of increased PI3K pathway activity. All patients demonstrated heterogeneity in phospho-AKT expression and phospho-AKT/pan-AKT ratio among individual CTCs at a single timepoint and across timepoints in longitudinal analysis. Conclusion: We report here the feasibility of quantitative and longitudinal detection of activated AKT in EpCAM/Trop2 captured CTCs from metastatic breast cancer with somatic PIK3CA mutations. Future work will prospectively evaluate multiple clinical applications of this assay in patients receiving alpelisib plus endocrine therapy for PIK3CA mutated HR+ metastatic breast cancer, as well as expanding the assay to include the detection of additional phospho-protein readouts of PI3K pathway activity in CTCs. In addition to the potential to complement solid biopsy PIK3CA mutation status as a predictive biomarker of sensitivity to PI3K therapies, this assay has the unique potential to provide a pharmacodynamic assessment of PI3K inhibitor activity in CTCs while on treatment, which may serve as an early biomarker of clinical response or resistance. Citation Format: Marina N Sharifi, Jamie M Sperger, Cole Gilsdorf, Serena K Wolfe, Amanda Parkes, Kari B Wisinski, Ruth M O'Regan, Joshua M Lang. Detection of PI3K pathway activation in circulating tumor cells in PIK3CA mutated metastatic breast cancer as a putative predictive biomarker for PI3K inhibitor therapies [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-35.

Abstract IA020: Chromatin regulation of the endometrium: What are our models telling us?

Abstract ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. ARID1A is a SWI/SNF chromatin-remodeling complex subunit. ARID1A loss in the mouse endometrial epithelium leads vaginal bleeding when combined with PI3K pathway activation. Sorted mutant endometrial epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT), inflammation and estrogen signaling in vivo. ARID1A loss leads to the expression of EMT genes, suggesting ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates. In contrast to its repressive roles, ARID1A is required for the activation estrogen responsive genes, including the progesterone receptor (PGR). Estrogen responsive genes are marked by bivalent chromatin domains containing both histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3). High H3K27me3 correlates with low PGR expression, suggesting Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 normally silences PGR. In support of this, pharmacological inhibition of PRC2 partially rescues PGR activation is estrogen treated, ARID1A-deficient cells. Given the known antagonistic relationship between SWI/SNF and PRC2 complexes, we propose that SWI/SNF normally opposes PRC2 mediated silencing of the PGR promoter in response to estrogen, leading to PGR expression. In the ARID1A mutant endometrium, PGR promoter silencing by PRC2 is maintained. In the absence of PGR, progesterone cannot oppose the actions estrogen in the endometrium, leading to unopposed estrogen signaling. Our data demonstrate ARID1A has both activating and repressive roles in the endometrium, and ARID1A loss contributes to multiple aspects of disease pathogenesis. Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Chromatin regulation of the endometrium: What are our models telling us? [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA020.

Treadmill exercise in obese maternal rats during pregnancy improves spatial memory through activation of phosphatidylinositol 3-kinase pathway in the hippocampus of rat pups.

Maternal nutrition is necessary for the growth of the fetus, and excessive intake of nutrients interferes with brain development in offspring. In the current study, the effect of treadmill running during pregnancy in obese maternal rats on spatial learning memory and spatial working memory in rat pups was investigated. Phosphorylation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and extracellular signal-regulated kinase 1 and 2 (ERK1/2) was also identified in rat pups. Female rats were divided into the normal diet group and the high-fat diet group for 7 weeks, including pregnancy and lactation. Maternal treadmill running was performed for 4 weeks. The born rat pups were classified into a control group, a treadmill exercise group, a high-fat diet group, a high-fat diet and treadmill exercise group according to the status of maternal rats. Radial 8-arm maze task for spatial learning memory and Morris water maze task for spatial working memory were done. Western blot analysis was conducted to determine the expressions of PI3K, Akt, ERK1/2. In the current results, maternal treadmill running during pregnancy improved spatial learning memory and spatial working memory in rat pups born to obese maternal rats. This improving effect of memory was due to the enhanced phosphorylation of PI3K, Akt, and ERK1/2 by treadmill running.

Stathmin as a surrogate marker of phosphatidylinositol-3-kinase pathway activity: Towards precision medicine in HPV-negative head & neck squamous cell carcinoma

In order to assess Stathmin as an immunohistochemical (IHC) indicator of phosphatidylinositol 3-kinase (PI3K) pathway activity in HPV-negative head & neck squamous cell carcinoma (HNSCC), we compared Stathmin IHC to expression of other pathway components. We also evaluated the relationship between Stathmin IHC and the mutational status of four key pathway genes. Finally, we ascertained whether Stathmin IHC correlates with tumor grade or primary site. Correlation exists between high Stathmin expression and high pAKT1 expression, indicating a role for Stathmin IHC as a marker of pathway activity. Our analysis did not show correlation between Stathmin IHC and mutation of the four genes evaluated. We also observed an association between high Stathmin expression and oropharyngeal primary site. Our results suggest utility of Stathmin IHC as an indicator of PI3K pathway activity, and thereby demonstrate potential relevance of Stathmin IHC in the context of HNSCC.

Scopoletin increases glucose uptake through activation of PI3K and AMPK signaling pathway and improves insulin sensitivity in 3T3-L1 cells

Coumarins have been shown to reduce blood glucose levels and improve insulin sensitivity in other studies. The purpose of this study was to investigate the effects of scopoletin, which is a type of coumarin family, on glucose uptake in 3T3-L1 cells to test the hypothesis that scopoletin exerts an antidiabetic function on adipocytes. Scopoletin significantly increased glucose uptake, which was associated with increased expression of the plasma membrane glucose transporter type 4 (PM-GLUT4) in 3T3-L1 adipocytes. This increase in PM-GLUT4 expression was promoted by phosphorylation of protein kinase B, activation of phosphatidylinositol-3-kinase (PI3K), and enhanced intracellular glucose uptake. Scopoletin also promoted phosphorylation of adenosine monophosphate–activated protein kinase (AMPK) and enhanced PM-GLUT4 expression. Scopoletin-induced glucose uptake in 3T3-L1 adipocytes was inhibited by treatment with the PI3K inhibitor wortmannin and the AMPK inhibitor compound C. These results suggest that scopoletin has an antidiabetic effect by stimulating GLUT4 translocation to the PM through activation of the PI3K and AMPK pathways in 3T3-L1 adipocytes, thereby upregulating glucose uptake.

Torin2 Exploits Replication and Checkpoint Vulnerabilities to Cause Death of PI3K-Activated Triple-Negative Breast Cancer Cells.

Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

CXCR4 Blockade By BL-8040 in T Cell Acute Lymphoblastic Leukemia Decreases Mitochondrial Mass and Induces Non-Apoptotic Cell Death

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. CXCL12 is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. We previously reported that BL-8040, a potent new CXCR4 antagonist with sustained receptor occupancy, is active as monotherapy against T-ALL in mice. Indeed, a 2-week course of daily BL-8040 resulted in a median reduction in tumor burden of 32.1-fold (range 6.8 to 176) across 5 different T-ALL xenografts. Preliminary data from a clinical trial of BL-8040 plus nelarabine for relapsed T-ALL also suggest therapeutic activity, with a complete remission rate observed in 4/8 patients (50%), which compares favorably to published response rates of approximately 30% with single agent nelarabine. Here, we explore molecular mechanisms by which CXCR4 blockade induces T-ALL death. NOD-scid IL2Rgammanull (NSG) mice were injected with P12-Ichikawa cells, a T-ALL cell line modified to express click beetle red luciferase and GFP. Following T-ALL engraftment, mice were treated with a single dose of BL-8040, and then leukemic cells in the bone marrow harvested 24-48 hours later. Treatment with BL-8040 resulted in a marked suppression of Akt and Erk1/2 phosphorylation, suggesting that signaling through CXCR4 is the major source of PI3 kinase pathway activation in T-ALL cells. Surprisingly, treatment with BL-8040 did not affect cellular proliferation, as measured by Ki67/FxCycle Violet staining or by EdU labeling. Moreover, no increase in apoptosis, as measured by annexin V or activated caspase 3 expression, was observed. These data suggest that CXCR4 blockade induces a non-apoptotic cell death. To explore this possibility further, we performed transcriptome sequencing on T-ALL cells recovered from mice 24 hours after 1 dose of BL-8040. A total of 151 differentially expressed genes (FDR of &lt; 0.05% and ≥ 2-fold change) were identified. Gene set enrichment analysis was strongly positive for alterations in oxidative phosphorylation, ribosome biogenesis, and carbohydrate metabolism. Ribosome function was assessed using O-propargyl-puromycin (OPP), which monitors global protein translation. No difference in global protein synthesis in T-ALL cells was observed after CXCR4 blockade in vivo. T-ALL cells are dependent on glutamine as a source of carbon, and PI3 kinase signaling positively regulates glutaminolysis. Thus, we hypothesized that CXCR4 blockade may induce T-ALL cell death by reducing glutamine metabolism. However, treatment of T-ALL cells in vitro with BL-8040 did not alter the cellular levels of glutamine or glutamate, as measured using a commercial bioluminescent assay. Confirmatory metabolic tracing studies using 13C-labeled glutamine and glucose are in progress. Finally, to explore the reduction in oxidative phosphorylation, we examined mitochondria function using Mitotracker Green. Treatment of T-ALL cells in vitro with BL-8040 for 24-48 hours induced a significant decrease in mitochondria number, suggesting induction of mitophagy. Collectively, these data suggest that T-ALL cells are addicted to CXCR4 signaling in vivo. CXCR4 blockade with BL-8040 induces a non-apoptotic cell death that is characterized by a loss of mitochondria. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership.

Efficacy and safety of buparlisib, a PI3K inhibitor, in patients with malignancies harboring a PI3K pathway activation: a phase 2, open-label, single-arm study.

Background: Phosphatidylinositol 3-kinase (PI3K) pathway activation plays a key role in tumorigenesis and has been associated with poor prognosis and resistance to multiple therapies in various cancers. Results: There were 146 patients enrolled; common tumor types were colorectal, sarcoma, and ovarian. Tumors had PI3K pathway alterations and a median of four mutations with tissue-specific patterns of mutation burden (lowest: sarcoma [2.5]; highest: esophagus, germ cell tumor, skin non-melanoma, vaginal [7]). The number of prior therapies did not correlate with the number of genetic alterations (Pearson r = –0.037). The clinical benefit rate was 15.1% (n = 22). An additional patient had an unconfirmed complete response. The most common adverse events were fatigue, nausea, hyperglycemia, decreased appetite, and diarrhea. Patient and Methods: In this phase 2, open-label, single-arm study, patients with solid or hematologic malignancies with PI3K pathway activation and progression on or after standard treatment received buparlisib (100 mg once daily). The primary endpoint was clinical benefit rate per local investigator assessment (response or stable disease at ≥16 weeks). Conclusions: Buparlisib was well tolerated, however efficacy was limited despite selection of PI3K pathway aberrations. Future studies may provide insight into buparlisib efficacy by refining the molecular selection of different tumor types.

1938P - Characterization of the mechanism of action and efficacy of MEN1611 (PA799), a novel PI3K inhibitor, in breast cancer preclinical models

BackgroundThe phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. Mutations in PIK3CA, the gene coding the p110α catalytic subunit of PI3K, are frequent in cancer and result in PI3K pathway activation. About 25% of HER2-positive breast cancers also carry PIK3CA mutations, known to confer resistance to anti-HER2 therapy. Here we report the preclinical characterization of MEN1611, a potent and selective orally available PI3K inhibitor, showing high activity against p110α mutant isoforms and sparing the δ isoform. MethodsIn vivo efficacy studies were evaluated through Tumour Volume Inhibition % (TVI%) and mRECIST criteria using cancer cell lines and patient-derived xenograft (PDX) models. Pathway activation and protein degradation analyses were performed by western blot and capillary electrophoresis immunoassay. Immune modulation was evaluated using murine macrophages differentiated in vitro. ResultsConsistent with its mechanism of action, MEN1611 down-modulates the PI3K/AKT signaling as well as selected downstream targets, both in vitro and in vivo. MEN1611 acts synergistically when combined with trastuzumab in several HER2-positive PIK3CA-mutated breast cancer cell lines and patient-derived xenograft models. The in vivo efficacy in trastuzumab-resistant models is supported by a long-lasting antitumor activity, paralleled by mechanistic down-regulation of pharmacodynamic biomarkers. Moreover, MEN1611 showed the ability to induce a dose-dependent alpha-isoform depletion, and to modulate the macrophage polarization towards a pro-inflammatory phenotype, consistent with its capability to co-inhibit P110γ. ConclusionsOverall, these preclinical data support the progression in the development of MEN1611 in breast cancer and pave the way to the B-Precise-01 clinical study, a multicentre phase Ib study. Clinical trial identificationNCT03767335. Legal entity responsible for the studyMenarini Ricerche S.p.A. FundingMenarini Ricerche S.p.A. DisclosureA. Fiascarelli: Full / Part-time employment: Menarini RIcerche S.p.A. G. Merlino: Full / Part-time employment: Menarini Ricerche S.p.A. S. Capano: Full / Part-time employment: Menarini Ricerche S.p.A. A. Paoli: Full / Part-time employment: Menarini Ricerche S.p.A.. A. Bressan: Full / Part-time employment: Menarini Ricerche. M. Bigioni: Full / Part-time employment: Menarini Ricerche S.p.A.. M. Scaltriti: Advisory / Consultancy: Memorial Sloan-Kettering Cancer Center. J. Arribas: Advisory / Consultancy: Vall d’Hebron Institute of Oncology. C. Bernadó Morales: Advisory / Consultancy: Vall d’Hebron Institute of Oncology. A. Pellacani: Leadership role, Full / Part-time employment: Menarini Ricerche S.p.A.. M. Salerno: Leadership role, Full / Part-time employment: Menarini Ricerche. M. Binaschi: Leadership role, Full / Part-time employment: Menarini Ricerche S.p.A.

PIPA: A phase Ib study of selective ß-isoform sparing phosphatidylinositol 3-kinase (PI3K) inhibitor taselisib (T) plus palbociclib (P) in patients (pts) with advanced solid cancers—Safety, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) analysis of the doublet combination.

3087 Background: Oncogenic hyperactivation of the PI3K pathway is common in multiple cancers, with preclinical data showing that CDK4/6 inhibitors sensitise PIK3CA mutant cancers to PI3K inhibitors. We report the activity of the P+T in solid tumors with PI3K pathway activation, along with the PD biomarker analysis. Methods: We previously reported the dose escalation phase identifying 125mg P given 3-weeks-on, 1-week-off in combination with T 2mg as the recommended phase 2 dose (R2PD, Lim, ASCO 2017). We report the results in solid tumors with confirmed activating mutations (mts) in the PI3K pathway, from dose escalation and expansion, with no prior exposure to CDK4/6 or PI3K pathway inhibitors. PD studies include analyses of platelet-rich plasma (PRP) and paired tumour biopsies. Results: 20 pts (median age 61, range 34-72) were treated at the doublet RP2D, M/F 7/13, with a median 4 prior treatments (range 2-11). Tumour types included colorectal, breast, lung, endometrial,oligodendroglioma and head and neck cancers. Durable disease control occurred in 3 patients with ER+ advanced breast cancer with responses lasting &gt;6 months including 1 pt with a H1047R PIK3CAmt with an ongoing RECIST PR&gt;36 cycles, 2 pts with PIK3CAmt colorectal cancer had RECIST SD for &gt;5 months, and 1 patient with a PIK3CGmt anaplastic oligodendroglioma had clinical and radiological benefit lasting 5.5 months. Treatment was well tolerated with predictable G1-2 adverse events (AEs). G3 toxicities of neutropenia (n=6), thrombocytopenia (2), rash (2), mucositis (1) and raised transaminases (1 each) were all transient with no G4/5 AEs. Significant decreases in tumour pRb, and pAKT and pGSK3ß in PRP, confirmed modulation of both CDK4/6 and PI3K pathways at R2PD. Conclusions: Doublet P+T is well tolerated at the combination RP2D, with PD evidence of PI3K and CDK4/6 modulation in both plasma and tumor. Promising preliminary anti-tumor activity is seen in a mixed histology cohort selected for activating PIK3 mutations. Clinical trial information: NCT02389842.

Adiponectin and Serine/Threonine Kinase Akt Modulation by Triiodothyronine and/or LY294002 in 3T3-L1 Adipocytes.

Adipose tissue (AT), an endocrine organ that modulates several physiological functions by synthesizing and releasing adipokines such as adiponectin, is a metabolic target of triiodothyronine (T3). T3 and adiponectin play important roles in controlling normal metabolic functions such as stimulation of fatty acid oxidation and increase in thermogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway is important for the differentiation of preadipocytes into adipocytes and can be activated by T3 for the transcription of specific genes, such as adiponectin. We examined the role of PI3K in adiponectin modulation by T3 action in murine adipocytes (3T3-L1). The 3T3-L1 adipocytes were treated with 1000 nM T3 for 1 h in the presence or absence of 50 μM LY294002 (LY), a PI3K inhibitor. Then, we assessed the expression of adiponectin and the phosphorylated serine/threonine kinase Akt (pAkt), a PI3K signaling protein, in the adipocytes. Adiponectin and pAKT levels were higher in the T3-adipocyte cells, whereas in the LY group adiponectin was elevated and pAKT was decreased compared to the control (C). PI3K pathway inhibition for 1 h and posterior treatment with T3, in LY + T3, reduced the adiponectin level and increased pAKT levels compared to those in LY. T3 stimulated adiponectin levels by PI3K pathway activation and T3 can compensate alteration in the PI3K pathway, because with inhibition of the pathway it is able to maintain the basal levels of adiponectin and pAKT.

Assessment of Functional Phosphatidylinositol 3-Kinase Pathway Activity in Cancer Tissue Using Forkhead Box-O Target Gene Expression in a Knowledge-Based Computational Model

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in cancer. Tumors are potentially sensitive to PI3K pathway inhibitors, but reliable diagnostic tests that assess functional PI3K activity are lacking. Because PI3K pathway activity negatively regulates forkhead box-O (FOXO) transcription factor activity, FOXO target gene expression is inversely correlated with PI3K activity. Aknowledge-based Bayesian computational model was developed to infer PI3K activity in cancer tissue samples from FOXO target gene mRNA levels and validated in cancer cell lines treated with PI3K inhibitors. However, applied to patient tissue samples, FOXO was often active in cancer types with expected active PI3K. SOD2 was differentially expressed between FOXO-active healthy and cancer tissue samples, indicating that cancer-associated cellular oxidative stress alternatively activated FOXO. To enable correct interpretation of active FOXO in cancer tissue, threshold levels for normal SOD2 expression in healthy tissue were defined above which FOXO activity is oxidative stress induced and below which PI3K regulated. In slow-growing luminal A breast cancer and low Gleason score prostate cancer, FOXO was active in a PI3K-regulated manner, indicating inactive PI3K. In aggressive luminal B, HER2, and basal breast cancer, FOXO was increasingly inactive or actively induced by oxidative stress, indicating PI3K activity. We provide a decision tree that facilitates functional PI3K pathway activity assessment in tissue samples from patients with cancer for therapy response prediction and prognosis.

Abstract 5802: Efficacy of PI3Kβ inhibitor AZD8186 in PTEN-deficient triple-negative breast cancer

Abstract Background: Phosphatase and tensin homologue (PTEN) loss is a frequently disrupted tumor suppressor in cancer. PTEN serves as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway to promote balanced cell proliferation, survival and differentiation. Consequently, loss of PTEN function increases cell proliferation, decreases cell death and contributes to tumor initiation. PTEN loss occurs across a variety of cancer subtypes, and these PTEN deficient tumors are dependent on PI3Kβ activity. As a result, therapeutic approaches that focus on the inhibition of PI3Kβ isoform in PTEN-null cells are of interest. We evaluate the efficacy of PI3Kβ inhibitors focused on triple negative breast cancers with loss of PTEN function. Methods: We studied the effects of p110β inhibitor AZD8186 in a panel of ten TNBC cell lines, which included four PTEN-mutant cell lines. Three of four cell lines were confirmed to have PTEN loss on western blot. In vitro, cell proliferation assays were performed to determine the half maximal inhibitory concentration (IC50) after 3 days of treatment and to test the effects in combination with chemotherapy. We confirmed the sensitivity of the PTEN-null cell lines to AZD8186 with colony formation assays. Western blot analysis was performed to assess PTEN expression and PI3K pathway activation in MDA-MB-436 and MDA-MB-468 cell lines, and to evaluate effects of AZD8186 on PI3K signaling. Results: Cell lines with PTEN loss were significantly more sensitive to AZD8186 in vitro (p= 0.008). AZD8186 inhibited PI3K signaling. Western blot revealed decreased activation of pAKT, pGSK3β, pPRAS40 and pS6. Activation of other pathways were evaluated with no activation of the MAPK/ERK or MEK pathways appreciated. AZD8186 treatment resulted in increased apoptosis but did not have a significant effect on cell-cycle progression in PTEN-deficient cell lines. AZD8186 was not synergistic with eribulin, paclitaxel or carboplatin; combinations with novel targeted therapies in vitro and in vivo are ongoing. Conclusion: AZD8186 had single agent efficacy in PTEN-deficient triple negative breast cancer cell lines in vitro. Further study is needed to identify rationale combinations and in vivo efficacy. Citation Format: Nicci Owusu-Brackett, Ming Zhao, Argun Akcakanat, Kurt W. Evans, Erkan Yuca, Funda Meric-Bernstam. Efficacy of PI3Kβ inhibitor AZD8186 in PTEN-deficient triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5802.