Targeting Phosphatidylinositol 3-kinase Research Articles

Rational drug design from phosphatidylinositol 3-kinase-α inhibitors through molecular docking and 3D-QSAR methodologies for cancer immunotherapy

Dysregulation or aberrant activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is commonly observed in various cancers and is associated with tumor growth, metastasis, and resistance to therapy. Targeting PI3K-α with appropriate inhibitors can disrupt this pathway, hindering cancer progression, and potentially enhancing the immune system’s ability to recognize and eliminate cancer cells. In this study, we aimed to design a novel and potent inhibitor of PI3K-α for cancer immunotherapy using rational drug design techniques, including virtual screening, molecular docking, and 3D-QSAR. We obtained the human PI3K-α protein (6PYS) complexed with (3S)-3-benzyl-3-methyl-5-[5-(2-methylpyrimidin-5-yl)pyrazolo[1,5-a]pyrimidin-3-yl]-1,3-dihydro-2H-indol-2-one (PJ5) from the RCSB Protein Data Bank. Virtual screening of ligands, integrated with predictive computational molecular docking and 3D-field-based-QSAR, was implemented using appropriate Schrödinger Maestro modules. Rational drug design was also carried out, and its clinical relevance was validated across several ADMET descriptors. Docking results suggested that a hybrid of sulfonamide and pyridine-based heterocyclic compounds, functionalized with potent moieties derived from alkaloids, exhibited adequate synergistic biological effects capable of enhancing sufficient biological activity against PI3K-α. A field-based 3D-QSAR model was built on four partial least squares factors, and five statistical metrics were employed to validate the model. The newly designed ligand from this approach, named 6’-amino-5’-(2-fluoro-1,3-oxazol-5-yl)-N-{[3-(hydroxymethyl)oxetan-3-yl]methyl}-3-methyl-[2,3’-bipyridine]-6-sulfonamide or T85, exhibited a predicted bioactivity (pIC50) of 8.25. The predicted ADMET properties of T85 fell reasonably within the range of recommended standards, especially adhering to Lipinski’s rule of five and Jorgensen’s rule of three. In conclusion, the results of this study offer significant insights into in silico drug design using a rational approach, which could expedite the discovery and development of new drug molecules.

A Phase II Study of Intermittent Duvelisib Dosing in Patients with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL)

Background. Targeting phosphatidylinositol 3-kinase (PI3K) has emerged as an efficacious approach for treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). However, toxicities of the PI3K inhibitors are an important concern limiting their use. Duvelisib is a potent oral inhibitor of both the PI3K-δ and PI3K-γ isoforms with proven efficacy in CLL/SLL. Here we report the results of a phase 2 study evaluating efficacy and safety of duvelisib with intermittent dosing in patients with relapsed/refractory CLL/SLL Methods. This open-label, single arm, phase 2 study enrolled patients with CLL/SLL treated with at least 1 prior line of therapy with active disease as defined by the iWCLL 2008 criteria. Duvelisib was given at a dose of 25 mg PO BID for 28-day cycles for a total of 12 weeks during the induction phase, followed by 75 mg PO BID 2 days on and 5 days off for repeated 28-day cycles during the maintenance phase until death, intolerance or disease progression. The primary endpoint was progression free survival (PFS) at 12 months; secondary endpoints were safety and clinical benefit. The trial was originally planned to enroll 27 patients but was stopped early due to difficulties with enrollment following changing FDA guidance for the use of PI3K inhibitors. Results. Fifteen patients were enrolled. Median age was 74 (59-82) years, 8/15 (53%) were female; patients were primarily Caucasian (13/15; 87%); performance status was 0-1, with 27% (4/15) ECOG 0 and 73% (11/15) ECOG 1. This was a heavily pre-treated patient population with a median of 3 prior lines of therapy (1-6). Among patients where results were available, 79% (11/14) had abnormal FISH results including 36% with TP53 gene loss, 27% del(13q), and 18% del(11q). Sixty-seven percent (8/12) had unmutated IGHV. Of the 15 patients treated, 4 were not evaluable for efficacy due to coming off study early for toxicity (2), development of MDS unrelated to study drug (1) or death due to complications from COVID-19 infection (1). Among the 11 efficacy-evaluable patients, 82% achieved PR, and 18% had stable disease. The 12-month PFS was 43% (95%CI: 18%-66%) and the median PFS was 9.9 months (95% CI: 5.5-NA). Among the 9 responders, the median duration of response (DOR) was 5.3 months (95%CI: 1.9-NA) and 12-month DOR was 22% (95%CI:3%-51%). Five patients (33%) discontinued therapy due to adverse events, which occurred during the induction phase in 4/5 of the patients. Five patients (33%) discontinued due to progressive disease - all during the maintenance phase of treatment. Additionally, 2 patients discontinued due to MD decision; one patient developed MDS which was unrelated to duvelisib; one patient died during treatment due to complications from COVID-19. Treatment was generally well tolerated with the most common grade 3 or higher adverse events being leukocytosis (20%), alanine aminotransferase (ALT) increase (13%), aspartate aminotransferase (AST) increase (13%), oral mucositis (13%) and colitis (13%). The adverse events leading to treatment discontinuation occurred primarily during induction. The most common adverse events of any grade included fatigue in 5 patients (33%), leukocytosis, vomiting, ALT increase, AST increase, diarrhea, and elevation of alkaline phosphatase, which occurred in 4 patients each (27%). Conclusions. We report the results of an open-label, phase 2 study utilizing a continuous dosing induction phase followed by an intermittent dosing maintenance phase of the PI3K inhibitor duvelisib in patients with relapsed/refractory CLL/SLL. Patients who received duvelisib on an intermittent schedule achieved some clinical benefit, however, the desired 12-month PFS goal of 50% was not reached. Adverse events in general were manageable and mainly related to gastrointestinal and liver toxicity, and patient died during treatment from complications from COVID-19 pneumonia. After an initial 12 weeks of continuous duvelisib treatment, the incorporation of intermittent dosing appears to be a viable option for patients with previously treated CLL/SLL.

Astrovirus replication is dependent on induction of double-membrane vesicles through a PI3K-dependent, LC3-independent pathway.

Human astrovirus is a positive-sense, single-stranded RNA virus. Astrovirus infection causes gastrointestinal symptoms and can lead to encephalitis in immunocompromised patients. Positive-strand RNA viruses typically utilize host intracellular membranes to form replication organelles, which are potential antiviral targets. Many of these replication organelles are double-membrane vesicles (DMVs). Here, we show that astrovirus infection leads to an increase in DMV formation through a replication-dependent mechanism that requires some early components of the autophagy machinery. Results indicate that the upstream class III phosphatidylinositol 3-kinase (PI3K) complex, but not LC3 conjugation machinery, is utilized in DMV formation. Both chemical and genetic inhibition of the PI3K complex lead to significant reduction in DMVs, as well as viral replication. Elucidating the role of autophagy machinery in DMV formation during astrovirus infection reveals a potential target for therapeutic intervention for immunocompromised patients. IMPORTANCE These studies provide critical new evidence that astrovirus replication requires formation of double-membrane vesicles, which utilize class III phosphatidylinositol 3-kinase (PI3K), but not LC3 conjugation autophagy machinery, for biogenesis. These results are consistent with replication mechanisms for other positive-sense RNA viruses suggesting that targeting PI3K could be a promising therapeutic option for not only astrovirus, but other positive-sense RNA virus infections.

First-in-human phase Ia study of the PI3Kα inhibitor CYH33 in patients with solid tumors

PIK3CA mutations are highly prevalent in solid tumors. Targeting phosphatidylinositol 3-kinase α is therefore an attractive strategy for treating cancers harboring PIK3CA mutations. Here, we report the results from a phase Ia, open label, dose-escalation and -expansion study (NCT03544905) of CYH33, a highly selective PI3Kα inhibitor, in advanced solid tumors. The primary outcomes were the safety, tolerability, maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of CYH33. The secondary outcomes included evaluation of pharmacokinetics, preliminary efficacy and changes in pharmacodynamic biomarkers in response to CYH33 treatment. The exploratory outcome was the relationship between the efficacy of CYH33 treatment and tumor biomarker status, including PIK3CA mutations. A total of 51 patients (19 in the dose escalation stage and 32 in the dose expansion stage) including 36 (70.6%) patients (4 in the dose escalation stage and 32 in the dose expansion stage) with PIK3CA mutations received CYH33 1–60 mg. The MTD of CYH33 was 40 mg once daily, which was also selected as the RP2D. The most common grade 3/4 treatment-related adverse events were hyperglycemia, rash, platelet count decreased, peripheral edema, and fatigue. Forty-two out of 51 patients were evaluable for response, the confirmed objective response rate was 11.9% (5/42). Among 36 patients harboring PIK3CA mutations, 28 patients were evaluable for response, the confirmed objective response rate was 14.3% (4/28). In conclusion, CYH33 exhibits a manageable safety profile and preliminary anti-tumor efficacy in solid tumors harboring PIK3CA mutations.

DDEL-08. NOVEL COMBINATION THERAPY USING PI3KINASE INHIBITION AND IMMUNE BASED THERAPY FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Abstract Hurdles to effective treatments for glioblastoma multiforme (GBM), the most aggressive form of brain cancer, are resistance to chemotherapy, radiation, and immune checkpoint inhibitors. GBMs, like other “difficult to treat” tumors, often over-utilize the phosphatidyl inositol 3 kinase (PI3K) pathway for survival, cell growth, and cell division, conferring both chemotherapy and radiation resistance. Furthermore, the PI3K pathway has been shown to modulate expression of Major Histocompatibility Complex (MHC) molecules, key components of immune recognition. Based on these observations, we examined the potential of a novel, small molecule PI3K inhibitor GCT.Glio.1 (NPT520-337), which was designed to cross the blood brain barrier, to increase the sensitivity of GBM to immune based therapies and radiation. We hypothesized that targeting PI3K would result in growth arrest and increase cell surface expression of MHC molecules and the PD-L1 checkpoint target. Treatment of GL261 mouse and human U251 GBM cell lines with GCT.Glio.1 caused growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1 and MHC class II, and synergized with radiation to cause growth arrest and cell death. Based on these results we designed and performed in vivo animal studies, implanting luciferase-flagged GL261 into the caudate nucleus of C57BL6 mice. We treated the animals orally with GCT.Glio.1 followed 24 hours later by anti-PD1 therapy in repeating cycles. The treatments significantly delayed progression of the tumor when compared to no treatment or either treatment alone, with several animals exhibiting complete regression as measured by MRI and bioluminescence. In conclusion, these results indicate that GCT.Glio.1 may sensitize GBM to immune checkpoint inhibitor and/or radiation therapies. Based on our encouraging pre-clinical data and early results from pre-clinical pharmacology and safety studies, we anticipate filing a pre-Investigational New Drug application (pre-IND) as the next step toward our goal of a clinical trial using this combination therapy.

Abstract LB136: Characterization of a novel PI3Kinase inhibitor for treatment of glioblastoma multiforme

Abstract The purpose of this study was to test the effects of a novel small molecule inhibitor of phosphatidylinositol-3 kinase (PI3K) for the treatment of glioblastoma multiforme (GBM). The key hurdles to effective treatment for GBM, the “most deadly” form of brain cancer, are resistance to chemotherapy, to radiation, and to immune check point inhibitor therapy. The overarching goal of our experiments has been to determine if a well-characterized small molecule inhibitor of PI3K, GCT.Glio.1, would improve treatment outcomes for GBM patients. The PI3K pathway has been shown to confer both chemotherapy and radiation resistance in GBM. GCT.Glio.1 (NPT520-337), a small molecule compound known to cross the blood brain barrier (BBB), was developed to target PI3K to treat GBM. GBMs, like other “difficult to treat” tumors, often over-express and actively utilize the PI3K pathway for survival, cell growth, and cell division. We used this small molecule inhibitor to determine how targeting the PI3K pathway impacts treatment resistance in GBM. We hypothesized that targeting PI3K would result in growth arrest, increase cell surface expression of a key immune check-point inhibitor target PD-L1, and would sensitize the GBM to treatment with radiation. Treatment of GL261 mouse GBM and U251 human GBM cell lines with GCT.Glio.1 resulted in profound growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1, and synergized with radiation to cause growth arrest and subsequent cell death. In conclusion, these results indicate that GCT.Glio.1 may be a strong candidate for treatment of GBM, and suggest that GCT.Glio.1 is a candidate for rationally designed combinations with currently available radiation and immune checkpoint inhibitor therapies. Currently, the animal studies using GCT.Glio.1 in the GL261 mouse model of GBM have been initiated. Based on our promising pre-clinical data and early encouraging results from pre-clinical pharmacology and safety studies, we anticipate filing a pre-Investigational New Drug application (pre-IND) as the next step toward our goal of a clinical trial using GCT.Glio.1 for GBM patients. Citation Format: Ekokobe Fonkem, M. Karen Newell-Rogers, Richard Tobin, Debbie Healey, Mita Das, Sara Bowen, Chad Quarles, John Clark. Characterization of a novel PI3Kinase inhibitor for treatment of glioblastoma multiforme [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 LB136.

Natural Compounds as Potential Regulators of the Phosphatidylinositol 3′ Kinase (PI3K) Pathway in Breast Cancer

Breast cancer is one of the most prevalent forms of cancer in women both globally and in India. Although breast cancer is characterized by different molecular subtypes, a majority of breast cancers appear to have mutations in the phosphatidylinositol 3′ kinase (PI3K)/ protein kinase B (Akt) pathway. Dysregulation of the PI3K/ Akt pathway in breast cancers plays important roles in promoting tumour growth, proliferation and invasion. Targeting PI3K mediated signalling cascades could be therefore of value for breast cancer treatment. Studies with synthetic inhibitors of the PI3K/ Akt pathway have yielded positive results but the efficacy shown by many of these inhibitors appear to be compromised by deleterious side effects. An alternative to syn-thetic inhibitors is the use of natural phytochemical compounds with anti-tumorigenic potential like apigenin, pomolic acid, resveratrol and its deriva-tives, curcumin, epigallocatechin-3 gallate and thymoquinone as potential inhibitors of PI3K/Akt signalling in breast cancer and such a strategy could lead to lesser side effects and a lower treatment cost. The current study ex-amines the importance of the PI3K pathway in breast cancer and discusses how regulation of aberrant signalling through this pathway by natural com-pounds could play an important role in breast cancer therapy.

Targeting PI3K Pathway in Pancreatic Ductal Adenocarcinoma: Rationale and Progress.

Simple SummaryPancreatic cancer has a dismal 5-year survival rate of 10%, making it one of the deadliest forms of malignancy. The poor prognosis associated with this disease is because it is resistant to almost every type of chemotherapy. Another major hallmark of pancreatic cancer is the presence of several activated oncogenic signaling pathways, including PI3K/Akt/mTOR, which promotes disease aggressiveness and therapeutic resistance. Previously, we have shown that targeted inhibition of PI3K/Akt/mTOR together led to a significant reduction of tumor burden and improvement of overall survival in an aggressive mouse model of pancreatic cancer. This review article discusses the significance of targeting PI3K and its direct downstream effector components in pancreatic cancer. Additionally, we will also update on the recent studies highlighting the tumor cell-extrinsic impact of PI3K inhibition in the modulation of the immune microenvironment within the context of this malignancy.Pancreatic ductal adenocarcinoma (PDAC) remains among the deadliest solid tumors that remain treatment-refractory and show a dismal prognosis. More than 90% of PDAC tumors harbor mutations in the K-Ras that exert a strong pro-tumorigenic effect by activating several downstream effector pathways, including phosphatidylinositol-3-kinase (PI3K)-Akt. The role of frequently activated PI3K/Akt pathway in promoting PDAC aggressiveness is well established. Therapeutic approaches targeting PI3K and downstream signaling components in different cellular compartments, including tumor, stromal and immune cells, have directly impacted the tumor burden in this cancer type. Our previous work has demonstrated that targeting the PI3K/Akt/mTOR pathway reduced tumor growth and improved survival in the genetic mouse model of PDAC. Here, we discuss the significance of targeting PI3K signaling and the biological impact of PI3K inhibition in modulating the tumor–stromal immune crosstalk within the microenvironment of pancreatic cancer. Furthermore, this review updates on the current challenges involving the therapeutic implications of targeting this pathway in PDAC.

Targeting PI3K and AMPKα Signaling Alone or in Combination to Enhance Radiosensitivity of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is characterized by poor survival. Radiotherapy plays an important role in treating TNBC. The purpose of this study was to determine whether inhibiting the AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K) pathways alone or in combination potentiates radiotherapy in TNBC. AMPKα1 and AMPKα2 knockdown diminished cyclin D1 expression and induced G1 cell cycle arrest but did not induce apoptosis alone or in combination with radiotherapy. Next, we analyzed the role of PI3K p85α, p85β, p110α, p110β, Akt1, and Akt2 proteins on TNBC cell cycle progression and apoptosis induction. Akt1 and p110α knockdown diminished cyclin D1 expression and induced apoptosis. Silencing Akt1 promoted synergistic apoptosis induction during radiotherapy and further reduced survival after radiation. Treatment with the Akt inhibitor, MK-2206 48 h after radiotherapy decreased Akt1 levels and potentiated radiation-induced apoptosis. Together, our results demonstrate that AMPKα, p110α, and Akt1 promote TNBC proliferation and that Akt1 is a key regulator of radiosensitivity in TNBC. Importantly, combining radiotherapy with the pharmacological inhibition of Akt1 expression is a potentially promising approach for the treatment of TNBC.

Wortmannin targeting phosphatidylinositol 3-kinase suppresses angiogenic factors in shear-stressed endothelial cells.

Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.

11C-Labeled Pictilisib (GDC-0941) as a Molecular Tracer Targeting Phosphatidylinositol 3-Kinase (PI3K) for Breast Cancer Imaging.

Pictilisib (GDC-0941) is an inhibitor of phosphatidylinositol 3-kinase (PI3K), part of a signaling cascade involved in breast cancer development. The purpose of this study was to evaluate the pharmacokinetics of pictilisib noninvasively by radiolabeling it with 11C and to assess the usability of the resulting [11C]-pictilisib as a positron-emission tomography (PET) tracer to screen for pictilisib-sensitive tumors. In this study, pictilisib was radiolabeled with [11C]-methyl iodide to obtain 11C-methylated pictilisib ([11C]-pictilisib) using an automated synthesis module with a high radiolabeling yield. Considerably higher uptake ratios were observed in MCF-7 (PIK3CA mutation, pictilisib-sensitive) cells than those in MDA-MB-231 (PIK3CA wild-type, pictilisib-insensitive) cells at all evaluated time points, indicating good in vitro binding of [11C]-pictilisib. Dynamic micro-PET scans in mice and biodistribution results showed that [11C]-pictilisib was mainly excreted via the hepatobiliary tract into the intestines. MCF-7 xenografts could be clearly visualized on the static micro-PET scans, while MDA-MB-231 tumors could not. Biodistribution results of two xenograft models showed significantly higher uptake and tumor-to-muscle ratios in the MCF-7 xenografts than those in MDA-MB-231 xenografts, exhibiting high in vivo targeting specificity. In conclusion, [11C]-pictilisib was first successfully prepared, and it exhibited good potential to identify pictilisib-sensitive tumors noninvasively, which may have a great impact in the treatment of cancers with an overactive PI3K/Akt/mTOR signal pathway. However, the high activity in hepatobiliary system and intestines needs to be addressed.

MiR-126-3p inhibits apoptosis and promotes proliferation by targeting phosphatidylinositol 3-kinase regulatory subunit 2 in porcine ovarian granulosa cells.

ObjectiveNumerous studies have indicated that the apoptosis and proliferation of granulosa cells (GCs) are closely related to the normal growth and development of follicles and ovaries. Previous evidence has suggested that miR-126-3p might get involved in the apoptosis and proliferation of GCs, and phosphatidylinositol 3-kinase regulatory subunit 2 (PIK3R2) gene has been predicted as one target of miR-126-3p. However, the molecular regulation of miR-126-3p on PIK3R2 and the effects of PIK3R2 on porcine GCs apoptosis and proliferation remain virtually unexplored.MethodsIn this study, using porcine GCs as a cellular model, luciferase report assay, mutation and deletion were applied to verify the targeting relationship between miR-126-3p and PIK3R2. Annexin-V/PI staining and 5-ethynyl-2′-deoxyuridine assay were applied to explore the effect of PIK3R2 on GCs apoptosis and proliferation, respectively. Real-time quantitative polymerase chain reaction and Western Blot were applied to explore the regulation of miR-126-3p on PIK3R2 expression.ResultsWe found that miR-126-3p targeted at PIK3R2 and inhibited its mRNA and protein expression. Knockdown of PIK3R2 significantly inhibited the apoptosis and promoted the proliferation of porcine GCs, and significantly down-regulated the mRNA expression of several key genes of PI3K pathway such as insulin-like growth factor 1 receptor (IGF1R), insulin receptor (INSR), pyruvate dehydrogenase kinase 1 (PDK1), and serine/threonine kinase 1 (AKT1).ConclusionMiR-126-3p might target and inhibit the mRNA and protein expressions of PIK3R2, thereby inhibiting GC apoptosis and promoting GC proliferation by down-regulating several key genes of the PI3K pathway, IGF1R, INSR, PDK1, and AKT1. These findings would provide great insight into further exploring the molecular regulation of miR-126-3p and PIK3R2 on the functions of GCs during the folliculogenesis in female mammals.

Therapeutic Potency of PI3K Pharmacological Inhibitors of Gastrointestinal Cancer.

Therapeutic targeting of phosphatidyl-inositol 3-kinase (PI3K) is considered as a possible strategy in several types of cancer, including gastrointestinal ones. In vitro and in vivo studies indicated the significance of proapoptotic and antiproliferative inhibition of PI3K. Although there are many phase 1 and 2 clinical trials on PI3K inhibitors in patients with gastrointestinal cancer, the molecular mechanism of PI3K targeting PI3K/ mTOR pathway is not clear. Panclass I, isoformselective, and dual PI3K/mTOR inhibitors are under investigation. This review aimed to indicate PI3K-dependent targeting mechanisms in gastrointestinal cancer and the evaluation of related clinical data.

Targeting Phosphatidylinositol 3-Kinase Signaling Pathway for Therapeutic Enhancement of Vascular-Targeted Photodynamic Therapy.

Vascular-targeted photodynamic therapy (PDT) selectively disrupts vascular function by inducing oxidative damages to the vasculature, particularly endothelial cells. Although effective tumor eradication and excellent safety profile are well demonstrated in both preclinical and clinical studies, incomplete vascular shutdown and angiogenesis are known to cause tumor recurrence after vascular-targeted PDT. We have explored therapeutic enhancement of vascular-targeted PDT with PI3K signaling pathway inhibitors because the activation of PI3K pathway was involved in promoting endothelial cell survival and proliferation after PDT. Here, three clinically relevant small-molecule inhibitors (BYL719, BKM120, and BEZ235) of the PI3K pathway were evaluated in combination with verteporfin-PDT. Although all three inhibitors were able to synergistically enhance PDT response in endothelial cells, PDT combined with dual PI3K/mTOR inhibitor BEZ235 exhibited the strongest synergism, followed in order by combinations with pan-PI3K inhibitor BKM120 and p110α isoform-selective inhibitor BYL719. Combination treatments of PDT and BEZ235 exhibited a cooperative inhibition of antiapoptotic Bcl-2 family protein Mcl-1 and induced more cell apoptosis than each treatment alone. In addition to increasing treatment lethality, BEZ235 combined with PDT effectively inhibited PI3K pathway activation and consequent endothelial cell proliferation after PDT alone, leading to a sustained growth inhibition. In the PC-3 prostate tumor model, combination treatments improved treatment outcomes by turning a temporary tumor regrowth delay induced by PDT alone to a more long-lasting treatment response. Our study strongly supports the combination of vascular-targeted PDT and PI3K pathway inhibitors, particularly mTOR inhibitors, for therapeutic enhancement. Mol Cancer Ther; 16(11); 2422-31. ©2017 AACR.

Abstract 392: Combined targeting of MET and PI3K improves efficacy in breast cancer models with concurrent MET/PI3K aberrations

Abstract Introduction: Detailed understanding of the genetic abnormalities that drive subsets of cancer has led to the development of highly specific inhibitors targeting key oncogenic pathways. Growing evidence implicates aberrations of both mesenchymal epithelial transition receptor (MET) and its key downstream phosphatidylinositol 3-kinase (PI3K) signaling with poorer prognosis in breast cancer. Co-targeting MET/PI3K and identifying mechanisms of response and resistance could ultimately inform a novel therapeutic strategy for breast cancer treatment. Based on these finding, we hypothesized that concurrent aberrations in PI3K and MET will render breast cancers resistant to therapies targeting each pathway, and that combination therapy targeting the PI3K and MET pathway will improve the efficacy by preventing the acquisition of resistance. Methods: We established cell models that stably express human wild type (WT) MET or MET-T1010I, a functional germline single nucleotide polymorphism, or mutant PI3K-H1047R alone, or variable concurrent aberrations in PI3K and MET using MCF-10A, a non-transformed mammary epithelial cell line, and HCC1954, a breast cancer cell line with an endogenous PI3KCA-H1047R mutation. Cell behaviors, including survival, invasion and response to MET one arm monoclonal antibody onartuzumab (MetMAb, Genentech Inc) and/or pan class I PI3K inhibitor GDC 941 (pictilisib, Genentech Inc), were detected. HCC1954 expressing WT MET or MET-T1010I along with PI3K-H1047R were transplanted into the mammary fat pad of hepatocyte growth factor-transgenic mice on a severe combined immunodeficiency background. Statistical analyses were carried out using the ANOVA and the Student t test. Result: Expression WT MET or MET-T1010I act coordinately with PI3K-H1047R to increase colony formation (P < 0.01, respectively). Concurrent MET-T1010I, but not WT MET, acts coordinately with PI3K-H1047R to increase cell invasion (P < 0.0001). Expression MET-T1010I or WT MET induced cells resistance to GDC941, with MET-T1010I stronger than WT MET. Combined targeting MET with onartuzumab and targeting PI3K with GDC941 significantly inhibited cell growth in matrigel, dose dependently than each monotherapy (P < 0.0001, respectively). Invasion assay showed similar pattern. Combined onartuzumab and GDC941 significantly inhibited invasion, dose dependently (P < 0.001). The effects were confirmed using PI3K/mTOR inhibitor, GDC980. Consistently, combination of onartuzumab/GDC941 significantly inhibited tumor growth (P < 0.01 versus GDC941; P < 0.0001 versus onartuzumab alone). Conclusions: Concurrent aberrations of MET and PI3K render breast cancer cells resistant to therapies blocking either pathway. Targeting both MET and PI3K increases inhibitory efficacy. Citation Format: Shuying Liu, Naoto T. Ueno, Bailiang Wang, Mihai Gagea, Prahlad T. Ram, Mark Merchant, John Mendelsohn, Debasish Tripathy, Gordon B. Mills, Ana M. Gonzalez-Angulo. Combined targeting of MET and PI3K improves efficacy in breast cancer models with concurrent MET/PI3K aberrations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 392.

Development of novel PET probes targeting phosphatidylinositol 3-kinase (PI3K) in tumors

Phosphatidylinositol 3-kinase (PI3K) activity and protein expression levels are often increased in tumor regions. Since PI3K plays a crucial role in regulating cell growth and proliferation, inhibiting PI3K-dependent pathways could be a promising approach for cancer treatment. In clinical practice, however, evaluation of PI3K expression levels is limited to immunohistochemistry of patient samples, which requires invasive biopsies.Here we report the synthesis of three candidate compounds, FMTA-1, 2 and 3, and evaluate their capacity to detect PI3K expression levels with positron emission tomography (PET). Among the three candidates, FMTA-2 showed a lower IC50 value for PI3K. 18F Radiolabeling of FMTA-2 to produce [18F]FMTA-2 was accomplished and its capacity for detecting PI3K expression levels was evaluated in vitro and in vivo. Cell uptake of [18F]FMTA-2 correlated well with cellular PI3K expression levels, and was suppressed by the ATP-competitive PI3K inhibitor ZSTK474. In an in vivo experiment using tumor-transplanted model mice, a higher signal-to-noise ratio (S/N) was seen with [18F]FMTA-2 in animals transplanted with DMS114 cells (expressing high PI3K levels) relative to DU145 cells (expressing low PI3K levels). However, in vivo pharmacokinetics of [18F]FMTA-2 was undesirable and the absolute amount of this compound that accumulated at the tumor region was low. To the best of our knowledge, this study represents the first trial of a PET tracer for detecting PI3K. Although further improvement of the probe is required prior to clinical application, these results should encourage future work.

Abstract 3800: Targeting PI3K signaling pathway for therapeutic enhancement of verteporfin-mediated photodynamic therapy

Abstract Photodynamic therapy (PDT) induces cell injury and death through generation of reactive oxygen species (ROS) after light activation. Verteporfin is a photosensitizer that has been approved for the treatment of age-related macular degeneration and is under investigation for targeting tumor vasculature for therapy. Being primarily localized in mitochondria, verteporfin-mediated PDT induced a rapid apoptotic cell death in SVEC mouse endothelial cells by activating mitochondria-initiated cell death pathways. However, activation of phosphatidylinositol 3-kinase (PI3K) signaling pathway was also observed after verteporfin-PDT, especially at a sub-lethal dose. Furthermore, activation of PI3K signaling was correlated with cell growth after PDT. Thus, the goal of this study is to test the hypothesis that therapeutic outcome of verteporfin-PDT can be enhanced by targeting PDT-induced pro-survival PI3K signaling pathway. We found that PI3K-mTOR pathway inhibitor NVP-BEZ235 (BEZ235) significantly inhibited PDT-induced PI3K signaling activation and combination of BEZ235 and verteporfin-PDT resulted in more cell apoptosis and greater inhibition in cell proliferation in SVEC cells. The combination therapy led to significantly more tumor growth inhibition than each individual treatment in the PC-3 prostate tumor model. Examination of tumor samples for PI3K signaling activity and functional vasculature revealed that the combination treatment caused more inhibition in PI3K signaling and reduction in functional blood vessels than verteporfin-PDT or BEZ235 treatment alone. These results indicate that targeting pro-survival PI3K signaling pathway is an effective approach for enhancing tumor response to verteporfin-PDT. Citation Format: Daniel E. Kraus, Pratheeba Palasuberniam, Bin Chen. Targeting PI3K signaling pathway for therapeutic enhancement of verteporfin-mediated photodynamic therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3800. doi:10.1158/1538-7445.AM2015-3800

Abstract B42: Targeting PI3K in personalized treatment of BRAF-mutated pediatric low-grade gliomas

Abstract Introduction: Pediatric low-grade gliomas (PLGG) constitute the most common group of central nervous system tumors in children. Although they exhibit rare malignant transformation and relatively slow growth, PLGGs pose great morbidity. Viewed as a chronic disease, ideal therapy for PLGG should carry limited side effects that can be best achieved through selective, personalized therapy. Despite the known heterogeneity of PLGGs and characterized driver mutations that together offer the perfect, timely platform for personalized approaches to therapy, most children are still treated with standard chemotherapy protocols in a “one treatment fits all” approach. Recent genomic studies have defined recurrent, diverse alterations driving WHO grade 1 and 2 PLGGs. Activation of mitogen protein kinase (MAPK) and phosphatidylinositol-3′ kinase (PI3K) in PLGGs suggests the potential efficacy of agents that target these key growth regulatory pathways. BRAF, a kinase within the MAPK pathway, is activated by missense mutation (V600E) in ∼20% of grade 2-4 pediatric gliomas or KIAA1549-BRAF fusion in ∼60% of pilocytic astrocytomas. In addition to RAS/MEK/MAPK/ERK, the PI3K/AKT/mTOR signaling cascade is activated in ∼half of PLGGs. We hypothesize that knowledge of individual tumor BRAF genotype can guide selection of PI3K/MAPK inhibitors as single agents and in combinations, to maximize efficacy of therapy and overcome innate and acquired resistance to targeted agents. Methods: We used human glioma cell lines containing BRAFV600E (AM38, DBT-RG, BT40), or wild-type BRAF (BRAFWT; SF188, SF9427, SF9402) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells and BRAFV600E-expressing murine brain tumors. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK). Cell cycle distribution and apoptosis were assessed using flow cytometry; proliferation was determined using an ATP-based assay (CellTiter-Glo). In vivo activity of these inhibitors was assessed in the BT40 PLGG xenograft mouse model. Results: In BRAFV600E cells, combination of PLX4720+everolimus and PLX4720+AZD6244 exhibited significantly greater effects on cell viability, apoptosis, and cell cycle than respective single agents. In BRAFWT cells, everolimus+AZD6244 was superior compared to respective monotherapies. Similar results were found using isogenic murine cells. In KIAA1549:BRAF fusion cells, MEK inhibition reduced cell viability and S-phase content, effects that were modestly augmented by mTOR inhibition. In vivo experiments in the PLGG xenograft model BT40 showed the greatest survival advantage in mice treated with combination of AZD6244+PLX4720 or AZD6244+everolimus compared with respective monotherapies (p<0.01). Conclusions: For BRAFV600E tumors, combination of everolimus+MEK or PLX4720+MEK inhibitors is equally efficacious and superior to single agents, and the choice between these two might be dictated by clinical tolerability. For BRAFWT PLGGs, combination of everolimus+MEK inhibitor is superior to single agent therapies. Optimal treatment of KIAA1549:BRAF-expressing PLGGs is still under investigation as single agent MEK inhibition is extremely efficacious but mTOR inhibition may contribute to greater overall anti-neoplastic effects. Citation Format: Aleksandra K. Olow, Sabine Mueller, Xiaodong Yang, Rintaro Hashizume, William Weiss, Adam C. Resnick, Angela J. Sievert, Mitchel S. Berger, Nalin Gupta, David C. James, Daphne A. Haas-Kogan. Targeting PI3K in personalized treatment of BRAF-mutated pediatric low-grade gliomas. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B42.

Abstract B53: Targeting PI3K signaling pathway for therapeutic enhancement of verteporfin-mediated photodynamic therapy

Abstract Photodynamic therapy (PDT) induces cell injury and death through generation of reactive oxygen species (ROS) after light activation. Verteporfin is a photosensitizer that has been approved for the treatment of age-related macular degeneration and is under investigation for targeting tumor vasculature for therapy. Being primarily localized in mitochondria, verteporfin-mediated PDT induced a rapid apoptotic cell death in SVEC mouse endothelial cells by activating mitochondria-initiated cell death pathways. However, activation of phosphatidylinositol 3-kinase (PI3K) signaling pathway was also observed after verteporfin-PDT, especially at a sub-lethal dose. Furthermore, activation of PI3K signaling was correlated with cell growth after PDT. Thus, the goal of this study is to test the hypothesis that therapeutic outcome of verteporfin-PDT can be enhanced by targeting PDT-induced pro-survival PI3K signaling pathway. We found that PI3K-mTOR pathway inhibitor NVP-BEZ235 (BEZ235) significantly inhibited PDT-induced PI3K signaling activation and combination of BEZ235 and verteporfin-PDT resulted in more cell apoptosis and greater inhibition in cell proliferation in SVEC cells. The combination therapy led to significantly more tumor growth inhibition than each individual treatment in the PC-3 prostate tumor model. Examination of tumor samples for PI3K signaling activity and functional vasculature revealed that the combination treatment caused more inhibition in PI3K signaling and reduction in functional blood vessels than verteporfin-PDT or BEZ235 treatment alone. These results indicate that targeting pro-survival PI3K signaling pathway is an effective approach for enhancing tumor response to verteporfin-PDT. Citation Format: Daniel Kraus, Bin Chen. Targeting PI3K signaling pathway for therapeutic enhancement of verteporfin-mediated photodynamic therapy. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B53.

Abstract IA13: Targeting PI3K isoforms in cancer: From mouse genetics to human therapy

Abstract The signaling axis of the class-I phosphatidylinositol 3-kinase (PI3K) is perhaps most commonly activated in cancer; activation of this pathway occurs via distinct mechanisms that include somatic activating mutations in PIK3CA (the gene encoding the p110α isoform of PI3K), genetic or epigenetic loss of the PTEN tumor suppressor and mutation/overexpression of RTKs. Thus PI3K represents an attractive target for cancer therapy and tremendous efforts are being devoted to developing effective PI3K inhibitors for cancer treatment. There are several PI3K isoforms in mammals, only the p110α and p110β isoforms are ubiquitously expressed. While the majority of the signal downstream of nearly all the RTKs and oncogenes so far tested is carried through p110α, p110β appears to play a prominent role in G protein mediated signaling in cells and for RTK signaling in certain circumstances. Surprisingly, we found that p110β is also the major player in a number of tumors driven by PTEN loss. In the mouse, each enzyme has been found to be necessary for certain processes and dispensable for others. Taking both genetic and pharmacological approaches, we have found that the two isoforms can actually play opposing roles in the normal development and pathology of mammary tissue. More recently, we found that p110α and p110β isoforms play spatially distinct roles in the epidermal skin that are required for the development and maintenance of PTEN hamartoma tumor syndrome. In this talk, I will discuss our most recent results on our studies of the distinct roles of the two isoforms in cancer. Citation Format: Jean J. Zhao. Targeting PI3K isoforms in cancer: From mouse genetics to human therapy. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA13.