pan-PI3K Inhibitor Research Articles

Validated HPLC-UV method for quantification of paxalisib, a pan PI3K and mTOR inhibitor in mouse plasma: Application to a pharmacokinetic study in mice.

Paxalisib is a pan-PI3K and mTOR inhibitor, currently entering into Phase II clinical trials as a potential drug to treat glioblastoma patients. We report the development and validation of a high-performance liquid chromatography (HPLC) method for the quantitation of paxalisib in mouse plasma as per the US Food and Drug Administration regulatory guidelines. From the mouse plasma, paxalisib and the internal standard (IS; filgotinib) were extracted using ethyl acetate as an extraction solvent. The chromatographic separation of paxalisib and the IS was accomplished on a Symmetry C18 (250×4.6mm, 5.0μm) column maintained at 40°C using 10mm ammonium formate and acetonitrile in gradient conditions at a 0.8ml/min flow-rate. The injection volume was 20 μl. The elution was monitored using a photo-diode array detector set at λmax 280 nm. Paxalisib and the IS eluted at 6.5 and 5.9min, respectively with a total run time of 10min. The calibration curve was linear over the range of 111-4,989 ng/ml. Inter- and intraday precision and accuracy, stability studies, dilution integrity and incurred sample reanalysis were investigated and the results met the acceptance criteria. The validated HPLC method was extended to assess the pharmacokinetic parameters of paxalisib in mice.

<i>PIK3CA</i>-mutated breast cancer: from research to clinical practice

Mutation or amplification in the PIK3CA gene is one of the most frequent somatic mutations in hormone-dependent Her2neu-negative breast cancer (HR+ Her2neu- breast cancer) and is associated with an increased risk of relapse, progression or death. Understanding the mechanisms leading to hyperactivation of the PI3K-mediated signaling cascade has led to a new class of drugs aimed at inhibiting/suppressing it. Studies of the efficacy of the pan-PI3K inhibitor buparlisib and the beta isoform inhibitor taselisib were formally positive and reached their planned primary sites, but were discontinued due to high levels of toxicity. To date, the only phosphatidylinositol 3-kinase (PI3K) inhibitor approved in clinical practice is alpelsib, which selectively inhibits the alpha isoform of the enzyme's catalytic subunit. The efficacy and safety of alpelisib in combination with fulvestrant for the treatment of postmenopausal women and men with HR+ HER2- PIK3CA-mutated advanced or metastatic breast cancer after prior therapy with aromatase inhibitors was demonstrated in the SOLAR-1 study. The next step proving the efficacy of combination therapy in patients previously treated with endocrine therapy in combination with CDK4/6 inhibitors was the open, multicenter, noncomparative three-arm study BYLieve. The current accumulated clinical experience confirms and complements the findings. In this article, we review clinical cases of the use of alpelisib in patients who previously received hormone therapy, including CDK4/6 inhibitors.

A pyridinesulfonamide derivative FD268 suppresses cell proliferation and induces apoptosis via inhibiting PI3K pathway in acute myeloid leukemia.

Aberration of PI3K signaling pathway has been confirmed to be associated with several hematological malignancies including acute myeloid leukemia (AML). FD268, a pyridinesulfonamide derivative characterized by the conjugation of 7-azaindole group, is a newly identified PI3K inhibitor showing high potent enzyme activity at nanomole concentration. In this study, we demonstrated that FD268 dose-dependently inhibits survival of AML cells with the efficacy superior to that of PI-103 (pan-PI3K inhibitor) and CAL-101 (selective PI3Kδ inhibitor) in the tested HL-60, MOLM-16, Mv-4-11, EOL-1 and KG-1 cell lines. Further mechanistic studies focused on HL-60 revealed that FD268 significantly inhibits the PI3K/Akt/mTOR signaling pathway, promotes the activation of pro-apoptotic protein Bad and downregulates the expression of anti-apoptotic protein Mcl-1, thus suppressing the cell proliferation and inducing caspase-3-dependent apoptosis. The bioinformatics analysis of the transcriptome sequencing data also indicated a potential involvement of the PI3K/Akt/mTOR pathway. These studies indicated that FD268 possesses high potent activity toward AML cells via inhibition of PI3K/Akt/mTOR signaling pathway, which sheds some light on the pyridinesulfonamide scaffold for further optimization and investigation.

MTOR inhibition amplifies the anti-lymphoma effect of PI3Kβ/δ blockage in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kβ/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell–like (ABC) and germinal center B-cell–like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kβ/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kβ/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kβ/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kβ/δ signaling and thus identifies a previously unappreciated role of the PI3Kβ isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kβ/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.

The NRF2 antagonist ML385 inhibits PI3K-mTOR signaling and growth of lung squamous cell carcinoma cells.

Lung squamous cell carcinoma (LUSC) currently has limited therapeutic options because of the relatively few validated targets and the lack of clinical drugs for some of these targets. Although NRF2/NFE2L2 pathway activation commonly occurs in LUSC, NRF2 has predominantly been studied in other cancer models. Here, we investigated the function of NRF2 in LUSC, including in organoid models, and we explored the activity of a small molecule NRF2 inhibitor ML385, which has not previously been investigated in LUSC. We first explored the role of NRF2 signaling in LUSC cancer cell line and organoid proliferation through NRF2 knockdown or ML385 treatment, both in vivo and in vitro. Next, we performed Western blot and immunofluorescence assays to determine the effect of NRF2 inhibition on PI3K-mTOR signaling. Finally, we used cell viability and clonogenic assays to explore whether ML385 could sensitize LUSC cancer cells to PI3K inhibitors. We find that downregulation of NRF2 signaling inhibited proliferation of LUSC cancer cell lines and organoids, both in vivo and in vitro. We also demonstrate that inhibition of NRF2 reduces PI3K-mTOR signaling, with two potential mechanisms being involved. Although NRF2 promotes AKT phosphorylation, it also acts downstream of AKT to increase RagD protein expression and recruitment of mTOR to lysosomes after amino acid stimulation. We also find that ML385 potentiates LUSC growth inhibition by a pan-PI3K inhibitor, which correlates with stronger inhibition of PI3K-mTOR signaling. Our data provide additional support for NRF2 promoting LUSC growth through PI3K-mTOR activation and support development of NRF2 inhibitors for the treatment of LUSC.

A phase II study of buparlisib in relapsed or refractory thymomas.

To investigate the efficacy and safety of buparlisib, an oral pan-PI3K inhibitor, in relapsed or refractory thymomas. This was a single center, single arm, open label phase II trial of buparlisib in patients with recurrent thymoma who have progressed after at least one prior line of treatment. The primary endpoint was objective response rate (complete response [CR] + partial response [PR]). Secondary endpoints included toxicity; progression free survival (PFS); overall survival (OS); disease control rate (DCR), i.e., the percentage of patients who achieve either PR or CR or stable disease [SD] for at least 4 months. Between 10/13/2014 and 1/18/2017, 14 patients with stage IV disease were enrolled. Median age was 58y (23-74). 71% were females and 71% white. All patients had WHO B2 (29%) or B3 (71%) thymoma. Patients received buparlisib for a median of 4.5m (2-33). At a median follow up of 16.6m (2.4-31.3), onr patients (7%) achieved a PR. DCR was 50%. Median PFS was 11.1m (95% CI 2.9 - 18.8). Median OS, updated as of March, 2021 was 22.5m (10.7-31.3). Most common grade 3-4 adverse events related to buparlisib were dyspnea (21%), rash (14%), elevated transaminases (14%), cough (7%), pneumonitis (7%), anxiety (7%), fatigue (7%) and hyperglycemia (7%). Reasons for treatment discontinuation included progression of disease (n= 5), rash (n=4), pulmonary toxicity (n=3), sinusitis (n=1), and disseminated toxoplasmosis plus autoimmune cholangitis (n=1). As of 3/2021, 8 patients have died, 7 due to disease progression and 1 due to central nervous system toxoplasmosis and autoimmune cholangitis. Buparlisib showed modest activity in patients with relapsed or refractory thymomas. Further investigation of PI3K pathway targeted therapy in thymoma is warranted. (clinicaltrials.gov ID: NCT02220855). clinicaltrials.gov, identifier (NCT02220855).

302 (PB082) - Improving responses to dual PI3K/BCL2 inhibition in lymphomas: results of a pharmacological screen with over 1,400 compounds

Background: Copanlisib, a pan-PI3K inhibitor with stronger activity against the alpha and delta isoforms, is Food and Drug Administration (FDA) approved for the treatment of patients with relapsed or refractory follicular lymphoma, and it is has shown clinical activity in other indolent lymphomas including marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphomas (Tarantelli Blood Adv 2020). A phase 1 study has explored this specific combination (NCT03886649), while others have explored similar schemes based on dual PI3K and BCL2 targeting.

Inhibition of Vps34 and p110δ PI3K Impairs Migration, Invasion and Three-Dimensional Spheroid Growth in Breast Cancer Cells.

Breast cancer is a heterogeneous disease that represents the most common cancer around the world; it comprises 12% of new cases according to the World Health Organization. Despite new approaches in early diagnosis and current treatment, breast cancer is still the leading cause of death for cancer mortality. New targeted therapies against key signalling transduction molecules are required. Phosphoinositide 3-kinase (PI3K) regulates multiple biological functions such as proliferation, survival, migration, and growth. It is well established that PI3K isoform-selective inhibitors show fewer toxic side effects compared to broad spectrum inhibition of PI3K (pan-PI3K inhibitors). Therefore, we tested the PI3K p110δ-selective inhibitor, IC87114, and Vps34-selective inhibitor, Vps34-IN1, on the breast cancer cell lines MCF-7 and MDA-MB-231, representing hormone-responsive and triple-negative breast cancer cells, respectively. Our data show that both inhibitors decreased migration of MCF-7 and MDA-MB-231 cells, and Vps34 also significantly impacted MCF-7 cell proliferation. Three-dimensional (3D) in vitro culture models show that IC87114 and Vps34-IN1 treatment reduced the growth of MCF-7 and MDA-MB-231 cells in 3D tumour spheroid cultures. This study identifies IC87114 and Vps34-IN1 as potential therapeutic approaches in breast cancer.

Current status of phosphoinotiside-3 kinase inhibitors in blood cancers.

Treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) underwent paradigm shifts, with targeted agents rapidly displacing chemotherapy. Phosphoinotiside-3 kinase (PI3K) is essential for survival and proliferation of neoplastic B cells and has proven a tractable target in NHL, with four agents receiving FDA approval in the last decade. This review summarizes key data and challenges associated with use of PI3K inhibitors in routine practice. Idelalisib and duvelisib are active in CLL and indolent NHL, including in patients with high-risk features. Despite differential targeting of PI3K isoforms, they exhibit comparable efficacy and adverse event profile including autoimmune events (transaminitis, colitis, pneumonitis), mediated by Treg/Th17 imbalance. Although copanlisib, a pan-PI3K inhibitor, is associated with a distinct safety profile (hyperglycemia, hypertension), preclinical studies indicate that umbralisib, a dual inhibitor of PI3Kδ and casein kinase 1ε, may have less effect on Tregs. However, both drugs may still cause immune-mediated toxicities. With close monitoring and management of adverse events, PI3K inhibitors continue to have a role in therapy of R/R CLL and NHL. Strategies to mitigate adverse events and increase efficacy of PI3K inhibitors include time-limited combination approaches, intermittent dosing schedules.

DHW-208, A Novel Phosphatidylinositol 3-Kinase (PI3K) Inhibitor, Has Anti-Hepatocellular Carcinoma Activity Through Promoting Apoptosis and Inhibiting Angiogenesis.

Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide with high prevalence and lethality. Due to insidious onset and lack of early symptoms, most HCC patients are diagnosed at advanced stages without adequate methods but systemic therapies. PI3K/AKT/mTOR signaling pathway plays a crucial role in the progression and development of HCC. Aberrant activation of PI3K/AKT/mTOR pathway is involved in diverse biological processes, including cell proliferation, apoptosis, migration, invasion and angiogenesis. Therefore, the development of PI3K-targeted inhibitors is of great significance for the treatment of HCC. DHW-208 is a novel 4-aminoquinazoline derivative pan-PI3K inhibitor. This study aimed to assess the therapeutic efficacy of DHW-208 in HCC and investigate its underlying mechanism. DHW-208 could inhibit the proliferation, migration, invasion and angiogenesis of HCC through the PI3K/AKT/mTOR signaling pathway in vitro. Consistent with the in vitro results, in vivo studies demonstrated that DHW-208 elicits an antitumor effect by inhibiting the PI3K/AKT/mTOR-signaling pathway with a high degree of safety in HCC. Therefore, DHW-208 is a candidate compound to be developed as a small molecule PI3K inhibitor for the treatment of HCC, and our study provides a certain theoretical basis for the treatment of HCC and the development of PI3K inhibitors.

Neutrophil Survival Signaling During Francisella tularensis Infection.

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.

Inhibiting PI3K-AKT-mTOR Signaling in Multiple Myeloma-Associated Mesenchymal Stem Cells Impedes the Proliferation of Multiple Myeloma Cells.

The microenvironment of cancer cells is receiving increasing attention as an important factor influencing the progression and prognosis of tumor diseases. In multiple myeloma (MM), a hematological cancer of plasma cells, mesenchymal stem cells (MSCs) represent an integral part of the bone marrow niche and tumor microenvironment. It has been described that MM cells alter MSCs in a way that MM-associated MSCs promote the proliferation and survival of MM cells. Yet, our understanding of the molecular mechanisms governing the interaction between MM cells and MSCs and whether this can be targeted for therapeutic interventions is limited. To identify potential molecular targets, we examined MSCs by RNA sequencing and Western blot analysis. We report that MSCs from MM patients with active disease (MM-Act-MSCs) show a distinct gene expression profile as compared with MSCs from patients with other (non-) malignant diseases (CTR-MSCs). Of note, we detected a significant enrichment of the PI3K–AKT–mTOR hallmark gene set in MM-Act-MSCs and further confirmed the increased levels of related proteins in these MSCs. Pictilisib, a pan-PI3K inhibitor, selectively reduced the proliferation of MM-Act-MSCs as compared with CTR-MSCs. Furthermore, pictilisib treatment impaired the MM-promoting function of MM-Act-MSCs. Our data thus provide a deeper insight into the molecular signature and function of MSCs associated with MM and show that targeting PI3K–AKT–mTOR signaling in MSCs may represent an additional therapeutic pathway in the treatment of MM patients.

PI3Kγ is a novel regulator of TNFα signaling in the human colon cell line HT29/B6

Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes phosphorylating phospholipids in the membrane, thereby, promoting the PI3K/AKT signaling cascade. PI3Ks are involved in a variety of fundamental cellular functions, including tumor necrosis factor α (TNFα)-induced tight junction (TJ) impairment-a hallmark of inflammatory bowel diseases. Most of the studies analyzing the role of class I PI3K signaling in epithelial barrier maintenance did not decipher which of the isoforms are responsible for the observed effects. By using wild-type and PI3Kγ-deficient HT-29/B6 cells, we characterized the functional role of PI3Kγ in these cells under inflammatory conditions. Measurement of the transepithelial electrical resistance and the paracellular flux of macromolecules revealed that monolayers of PI3Kγ-deficient cells, compared with wild-type cells, were protected against TNFα-induced barrier dysfunction. This effect was independent of any PI3K activity because treatment with a pan-PI3K inhibitor did not alter this observation. By immunostaining, we found correlative changes in the distribution of the TJ marker ZO-1. Furthermore, the absence of PI3Kγ reduced the basal level of the pore-forming TJ protein claudin-2. Our study suggests a novel noncanonical, kinase-independent scaffolding function of PI3Kγ in TNFα-induced barrier dysfunction.

Abstract 2660: Targeting PI3K pathway to reprogram the tumor immune niche in angiosarcoma

Abstract Angiosarcoma is an aggressive, albeit rare, cancer in humans. Angiosarcomas are vascular malignancies that can occur anywhere in the body, and their metastatic propensity is high. The vascular sarcomas consist of disorganized, malignant vessel-forming cells. Recurrent mutations in the PIK3CA gene have been identified in angiosarcomas, and activation of the PI3K pathway appears to establish angiogenic programs. Naturally occurring hemangiosarcoma in dogs shares clinical and pathological features with human angiosarcoma. Our previous work has revealed that canine hemangiosarcomas and human angiosarcomas share mutational and transcriptional signatures that activate convergent PI3K signaling pathways. Appropriately powered canine studies take a comparative genomics approach, informing our research into human angiosarcomas. The cause of most sporadic angiosarcomas is unknown. Moreover, no therapeutic targets have been identified to improve outcomes. This study hypothesizes that the activation of PIK3CA mutations contributes to the molecular programs that modulate the immune niche in angiosarcoma. We first induced PIK3CA hotspot mutations (H1047R) in canine hemangiosarcoma cell line DHSA-1426 using CRISPR/Cas9. We found that PI3K-alpha selective inhibitor BYL719 at 10uM decreased cell proliferation of PIK3CA mutant cells by 40-50% compared with that of wild-type cells. In comparison, no significant difference was observed between mutant and wild-type cells treated with pan PI3K inhibitor, ZSTK474. Our western blot data showed an increase in γH2AX, a marker of DNA damage, in mutant cells when treated with BYL719 in a dose-dependent manner. BYL719 inhibited the phosphorylation of AKT and ERK in PIK3CA mutant tumor cells. Our RNA-seq transcriptomic data analysis revealed that PIK3CA mutations altered gene regulation programs associated with lipid biosynthesis, including phosphatidylinositol, DNA replication, angiogenic and vascular functions, and immune regulation. These data suggest that the activation of the PI3K pathway is essential for tumor cell growth and that PI3K-alpha selective inhibitor interferes with PIK3CA mutation-induced pathways, including AKT and ERK, through DNA damage-dependent mechanism in angiosarcoma. Our ongoing work is to determine if PIK3CA mutant tumor cells have cell-autonomous capacity to govern hematopoietic progenitors and immune cells, potentially establishing the tumor immune niche. It also includes establishing single-cell gene expression profiles to identify distinct cell clusters between PIK3CA mutant and wild-type cells, along with chromatin accessibility landscape using ATAC-seq. Tumorigenic capacity and immune regulatory mechanisms of PIK3CA mutations will be determined in angiosarcoma xenograft models. Citation Format: Emma Kozurek, Zhiyan Silvia Liu, Hai Dang Nguyen, Jong Hyuk Kim. Targeting PI3K pathway to reprogram the tumor immune niche in angiosarcoma [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 2660.

Abstract 3251: Aurora kinase B expression shields HNSCC from PI3K inhibition-induced apoptosis through downstream mediators AKT and PDK1

Abstract Effective targeted therapies are lacking for head and neck squamous cell carcinoma (HNSCC) that is common and fatal. Developing targeted therapies for HNSCC is challenging as the genomic landscape is dominated by mutations in tumor suppressors, including NOTCH1. To address this need, we previously demonstrated that PI3K inhibition led to apoptosis in NOTCH1 mutant HNSCC cell lines. The underlying mechanism of this sensitivity is unknown and important to elucidate as modest responses and development of acquired resistance are the leading causes of failure for targeted therapies. To address this knowledge gap, we measured the levels of 304 proteins using reverse phase protein array in NOTCH1WT and NOTCH1MUT HNSCC cell lines upon PI3K inhibition and identified Aurora kinase B (AURKB) to be differentially regulated. We also identified AURKB levels to be maintained in the NOTCH1MUT cell lines with acquired resistance to PI3K inhibition. To determine if the maintenance of AURKB expression contributes to PI3K inhibitor resistance, we depleted AURKB in resistant NOTCH1WT HNSCC cells and overexpressed AURKB in sensitive NOTCH1MUT HNSCC cells. This manipulation of AURKB levels led to increased sensitivity and resistance to PI3K inhibition respectively. To use a pharmacologic approach, we combined the pan-Aurora kinase inhibitor danusertib (0-2µM) with PI3K/mTOR inhibitor omipalisib (0-200nM) in 56 HNSCC cell lines for 72h and observed a substantial decrease in cell viability in >80% of NOTCH1WT and >90% of NOTCH1MUT HNSCC lines. Additionally, concurrent Aurora kinase and PI3K inhibition in NOTCH1WT, NOTCH1MUT, and NOTCH1MUT acquired resistant HNSCC cells for 24h resulted in elevated cell death compared to single agents, as measured by the induction of cleaved PARP and cleaved Caspase 3; and Annexin V positive cells. Mice bearing NOTCH1MUT xenografts showed complete tumor regression when treated with a combination of pan-PI3K inhibitor Copanlisib and Aurora inhibitor Alisertib as compared to control groups. AURKB depletion in NOTCH1WT and overexpression in NOTCH1MUT HNSCC cells revealed significant changes in the total protein levels of AKT and PDK1. Similarly, AKT depletion and overexpression in NOTCH1WT and NOTCH1MUT HNSCC cells altered sensitivity to PI3K inhibition. However, manipulation of AKT levels affected PDK1 and not AURKB levels, demonstrating AURKB to be upstream to AKT and PDK1. Therefore, maintenance of AURKB levels mediates resistance to PI3K inhibition in HNSCC through AKT and PDK1. We identified AURKB as a central player governing the sensitivity to PI3K inhibitor-induced apoptosis in the context of NOTCH1 mutation status in HNSCC through its effects on AKT and PDK1. These novel findings may lead to the development of more robust therapeutic approach for NOTCH1 mutant squamous carcinoma as well as patients who develop acquired resistance to targeted therapies. Citation Format: Pooja A. Shah, Anne M. Fernandez, Vaishnavi Sambandam, Hongyun Zhao, Tuhina Mazumdar, Li Shen, Jing Wang, Faye M. Johnson. Aurora kinase B expression shields HNSCC from PI3K inhibition-induced apoptosis through downstream mediators AKT and PDK1 [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 3251.

Precision medicine: Novel PI3K inhibitor as cancer treatment.

e15093 Background: Phosphoinositide 3-kinase (PI3K) is an attractive anti-cancer drug target due to its promotion of cell growth and proliferation; however, available pan-PI3K inhibitors currently lack specificity, leading to excessive drug toxicity and adverse effects. Selectide-18 is a novel PI3K inhibitor that targets PI3K’s catalytic subunit p110β, resulting in reduced viability of cancer cells. The p110β subunit is the most prominent PI3K subunit in many chemoresistant tumors. Previous research has revealed a unique 18-residue long motif within the C2 domain of p110β. This motif is not found in other catalytic subunits (p110α, p110δ, or p110γ), making it a promising drug target. Selectide-18 is a memetic peptide drug modeled after this motif and has been shown to effectively inhibit p110β binding without affecting other isoforms, separating it from currently available pan-PI3K inhibitors. While Selectide-18 has been successful in inhibiting PI3K in previous studies, it has poor cell permeability due to its large size. Previous findings have revealed functional redundancies among Selectide-18’s residues. This study aims to remove these redundancies and determine the optimal formulation of Selectide-18 through in silico analysis. Methods: An in silico protocol was developed to determine the optimal formulation of Selectide-18. By utilizing the PEP-FOLD server, 455 peptide models were created using 91 unique fragments of the Selectide-18 amino acid sequence ranging in size from 6 to 17 residues. Physical conformation of each model was compared to wild-type Selectide-18 using root-mean-square deviation data collected using PyMOL and UCSF Chimera software. Computational docking through AutoDock Vina was performed to test the binding affinity of each model to the active site of regulatory subunit p85α. The distance between each drug and the active site was measured with UCSF Chimera’s distance function. Finally, ClusPro docking of drug-bound p85α/p110β was compared to wild-type p85α/p110β, as well as non-truncated Selectide-18-bound p85α/p110β. Statistical significance was determined using a Student's t-test. Results: Select truncated models demonstrated physical confirmation (RMSD = 0.024), binding affinity (-8.74 kcal/mol), and simulated inhibitory capabilities comparable to Selectide-18. A specific group of variants comprised of 9 amino acids (9 residue_6K-14Q) had the shortest formulation without significant declines in binding affinity. Conclusions: Selectide-18 is a selective PI3K inhibitor that can be optimized by removing functionally redundant amino acids. A 9-residue variant ranging from amino acids 6Lys to 14Gln showed the most promise and will be used in future studies to reformulate Selectide-18. Being half the size of Selectide-18, this optimized drug is anticipated to have improved cell permeability without sacrificing inhibitory function.

Combining radiation with PI3K isoform-selective inhibitor administration increases radiosensitivity and suppresses tumor growth in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a malignant lung tumor with a dismal prognosis. The activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is common in many tumor types including NSCLC, which results in radioresistance and changes in the tumor microenvironment. Although pan-PI3K inhibitors have been tested in clinical trials to overcome radioresistance, concerns regarding their excessive side effects led to the consideration of selective inhibition of PI3K isoforms. In this study, we assessed whether combining radiation with the administration of the PI3K isoform-selective inhibitors reduces radioresistance and tumor growth in NSCLC. Inhibition of the PI3K/AKT pathway enhanced radiosensitivity substantially, and PI3K-α inhibitor showed superior radiosensitizing effect similar to PI3K pan-inhibitor, both in vitro and in vivo. Additionally, a significant increase in DNA double-strand breaks (DSB) and a decrease in migration ability were observed. Our study revealed that combining radiation and the PI3K-α isoform improved radiosensitivity that resulted in a significant delay in tumor growth and improved survival rate.

Patient-derived models of brain metastases recapitulate human disseminated disease

SummaryDissemination of cancer cells from primary tumors to the brain occurs in many cancer patients, increasing morbidity and death. There is an unmet medical need to develop translational platforms to evaluate therapeutic responses. Toward this goal, we established a library of 23 patient-derived xenografts (PDXs) of brain metastases (BMs) from eight distinct primary tumors. In vivo tumor formation correlates with patients’ poor survival. Mouse subcutaneous xenografts develop spontaneous metastases and intracardiac PDXs increase dissemination to the CNS, both models mimicking the dissemination pattern of the donor patient. We test the FDA-approved drugs buparlisib (pan-PI3K inhibitor) and everolimus (mTOR inhibitor) and show their efficacy in treating our models. Finally, we show by RNA sequencing that human BMs and their matched PDXs have similar transcriptional profiles. Overall, these models of BMs recapitulate the biology of human metastatic disease and can be valuable translational platforms for precision medicine.

PI3K Inhibitors in Advanced Breast Cancer: The Past, The Present, New Challenges and Future Perspectives.

Simple SummaryMolecular aberrations in the phosphatidylinositol 3-kinase (PI3K) pathway are often observed in breast cancer and represent a key regulator of many cellular processes, promoting tumor cell growth and survival. The first clinical trials leading to the development of pan-PI3K inhibitors showed certain preclinical activity; nevertheless, the toxicity profile limited further analysis of this drugs’ class. To improve the antitumor effect and therapeutic index, additional clinical trials have been performed to develop the PI3K isoform-specific inhibitors and new schedule combinations with a good toxicity profile. However, further efforts are needed to discover other potentially actionable genetic alterations that remain a challenge to reach the goal of personalized and precision medicine.Breast cancer is the leading cause of death in the female population and despite significant efforts made in diagnostic approaches and treatment strategies adopted for advanced breast cancer, the disease still remains incurable. Therefore, development of more effective systemic treatments constitutes a crucial need. Recently, several clinical trials were performed to find innovative predictive biomarkers and to improve the outcome of metastatic breast cancer through innovative therapeutic algorithms. In the pathogenesis of breast cancer, the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/AKT)-mammalian target of rapamycin (mTOR) axis is a key regulator of cell proliferation, growth, survival, metabolism, and motility, making it an interest and therapeutic target. Nevertheless, the PI3K/AKT/mTOR cascade includes a complex network of biological events, needing more sophisticated approaches for their use in cancer treatment. In this review, we described the rationale for targeting the PI3K pathway, the development of PI3K inhibitors and the future treatment directions of different breast cancer subtypes in the metastatic setting.

The Inhibitory Response to PI3K/AKT Pathway Inhibitors MK-2206 and Buparlisib Is Related to Genetic Differences in Pancreatic Ductal Adenocarcinoma Cell Lines.

The aberrant activation of the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) pathway is common in pancreatic ductal adenocarcinomas (PDAC). The application of inhibitors against PI3K and AKT has been considered as a therapeutic option. We investigated PDAC cell lines exposed to increasing concentrations of MK-2206 (an AKT1/2/3 inhibitor) and Buparlisib (a pan-PI3K inhibitor). Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated. Further, whole-exome sequencing (WES) and RNA sequencing (RNA-seq) were performed to analyze the recurrent aberrations and expression profiles of the inhibitor target genes and the genes frequently mutated in PDAC (Kirsten rat sarcoma virus (KRAS), Tumor protein p53 (TP53)). MK-2206 and Buparlisib demonstrated pronounced cytotoxic effects and limited cell-line-specific effects in cell death induction. WES revealed two sequence variants within the direct target genes (PIK3CA c.1143C > G in Colo357 and PIK3CD c.2480C > G in Capan-1), but a direct link to the Buparlisib response was not observed. RNA-seq demonstrated that the expression level of the inhibitor target genes did not affect the efficacy of the corresponding inhibitors. Moreover, increased resistance to MK-2206 was observed in the analyzed cell lines carrying a KRAS variant. Further, increased resistance to both inhibitors was observed in SU.86.86 carrying two TP53 missense variants. Additionally, the presence of the PIK3CA c.1143C > G in KRAS-variant-carrying cell lines was observed to correlate with increased sensitivity to Buparlisib. In conclusion, the present study reveals the distinct antitumor effects of PI3K/AKT pathway inhibitors against PDAC cell lines. Aberrations in specific target genes, as well as KRAS and TP53, individually or together, affect the efficacy of the two PI3K/AKT pathway inhibitors.