PI3Kα Signaling Research Articles

Abstract 1955: Novel long-acting covalent PI3Kα inhibitors boost potential action in cancer

Abstract Phosphoinositide 3-kinase (PI3K) is a key regulator of cell proliferation, survival, and metabolism. Hotspot mutations in the PIK3CA gene constitutively activate the catalytic subunit of the PI3Kα isoform and are found in 10-30% of solid tumors. The development of PI3K pathway inhibitors has been hampered by poor drug tolerance and release of negative feedback loops, leading to rapid reactivation of signaling in response to reversible inhibitors. To remedy these challenges, we developed highly selective covalent PI3Kα inhibitors that irreversibly bind to a non-conserved cysteine (C862) located 11 Å outside the ATP-binding site. Here we present the properties of an optimized drug-like covalent lead, dubbed compound 9, and describe the generation of a biotinylated probe developed to be used in conjunction with compound 9 to monitor PI3Kα target occupancy in relation to downstream PI3K signaling outputs. Biochemical investigations of compound 9 using TR-FRET assays showed high affinity binding (Ki), high reaction rates (kinact) for covalent bond formation with PI3Kα, and negligible off-target reactivity. Cellular NanoBRET assays revealed that compound 9 diffuses three times more rapidly into cells as compared to BYL719 (alpelisib), and leads to rapid and potent on-target engagement. Covalent bond formation to residue C862 of PI3Kα was further confirmed by X-ray crystallography. In PIK3CA mutant cancer cell lines, covalent binding of PI3Kα occurred at low nanomolar concentrations and led to persistent inhibition of Akt/PKB phosphorylation for more than 72 hours after drug removal. Notably, investigation of PI3Kα re-synthesis after labeling with compound 9 showed a negligible reappearance in cancer cell lines, which indicates that persistent suppression of PI3Kα signaling by compound 9 results in a much longer half-life for PI3Kα than previously reported. The irreversible inactivation of PI3Kα by compound 9 provides a marked gain in growth inhibition potency (10-600-fold) in PIK3CA mutant cancer cell lines in comparison to clinical reversible PI3Kα inhibitors and allows for intermittent dosing without compromising efficacy. These data collectively demonstrate that compound 9 enables precise and selective targeting of PI3Kα with high potency and long-lasting efficacy in cancer models. The new class of irreversible PI3Kα inhibitors have a unique pharmacology among PI3K inhibitors characterized by strong decoupling of drug exposure from efficacy which may provide opportunities to lower treatment burden in patients. Citation Format: Theodora A. Constantin, Lukas Bissegger, Erhan Keleş, Luka Raguž, Clara Orbegozo, Thorsten Schaefer, Isobel Barlow-Busch, John E. Burke, Chiara Borsari, Matthias P. Wymann. Novel long-acting covalent PI3Kα inhibitors boost potential action in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1955.

Identification of novel curcumin derivatives against pancreatic cancer: a comprehensive approach integrating 3D-QSAR pharmacophore modeling, virtual screening, and molecular dynamics simulations

Pancreatic cancer, known as the “silent killer,” poses a daunting challenge in cancer therapy. The dysregulation of the PI3Kα signaling pathway in pancreatic cancer has attracted considerable interest as a promising target for therapeutic intervention. In this regard, the use of curcumin derivatives as inhibitors of PI3Kα has emerged, providing a novel and promising avenue for developing effective treatments for this devastating disease. Computational approaches were employed to explore this potential and investigate 58 curcumin derivatives with cytotoxic activity against the Panc-1 cell line. Our approach involved ligand-based pharmacophore modeling and atom-based 3D-QSAR analysis. The resulting QSAR model derived from the best-fitted pharmacophore hypothesis (AAHRR_1) demonstrated remarkable performance with high correlation coefficients (R2) of 0.990 for the training set and 0.977 for the test set. The cross-validation coefficient (Q2) of 0.971 also validated the model’s predictive power. Tropsha’s recommended criteria, including the Y-randomization test, were employed to ensure its reliability. Furthermore, an enrichment study was conducted to evaluate the model’s performance in identifying active compounds. AAHRR_1 was used to screen a curated PubChem database of curcumin-related compounds. Two molecules (CID156189304 and CID154728220) exhibited promising pharmacokinetic properties and higher docking scores than Alpelisib, warranting further investigation. Extensive molecular dynamics simulations provided crucial insights into the conformational dynamics within the binding site, validating their stability and behavior. These findings contribute to our understanding of the potential therapeutic effectiveness of these compounds as PI3Kα inhibitors in pancreatic cancer. Communicated by Ramaswamy H. Sarma

Abstract 4945: Inhibition of wild-type PI3Kα signaling is required for durable efficacy in PI3Kα mutant cancer cells due to robust re-activation of wild-type PI3Kα signaling

Abstract PI3Kα is the most mutated oncogene with a high mutation rate in breast, lung, colorectal, gastric, bladder, and other tumor types. However, current inhibitors have had little efficacy in the clinic due to their inability to achieve maximal PI3Kα target engagement in a highly specific and durable manner. Contributing to this is that most PI3Kα mutant cancer cells are prone to robust pathway feedback and re-activation of remaining wild type PI3Kα signaling upon initial inhibition. We have analyzed pathway feedback in a variety of PI3Kα mutant cancer cell lines across multiple diverse indications and found this to be a common trait among PI3Kα mutant cancer cells. Additionally, when the mutant PI3Kα protein alone is targeted in these cells using mutant-specific inhibitors, we observed robust pathway re-activation and tumor cell proliferation. In contrast, TOS-358, a highly selective first-in-class covalent PI3Kα inhibitor, achieved complete inhibition of both PI3Kα mutant and wild type signaling and this correlated with the induction of cell cycle arrest and cell death across a wide variety of PI3Kα mutant tumor types. Furthermore, stimulation of PI3Kα signaling with serum rapidly re-activated the PI3K-AKT pathway in the presence of reversible and mutant specific inhibitors. Covalent molecules (namely TOS-358) achieved complete inhibition of pathway signaling in this format. Overall, this reveals the need to achieve sustained inhibition of both PI3Kα mutant and wild type signaling to achieve durable efficacy in PI3Kα mutant tumors. Citation Format: John R. MacDougall, Mengqi Zhong, Hanne Merritt, Joselyn S. Del Cid, John Bradley, Raymond Mak, Neil Dhawan, Wei Chen. Inhibition of wild-type PI3Kα signaling is required for durable efficacy in PI3Kα mutant cancer cells due to robust re-activation of wild-type PI3Kα signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4945.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin β-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy

Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells co‐ordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient‐rich conditions, is unknown. Here, we identify an endosome‐dependent phosphoinositide pathway that links PI3Kα signaling to lysosome repopulation during basal autophagy. We show that PI3Kα‐derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvation‐induced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5‐kinase, PIKfyve, to generate PI(3,5)P2. The SNX‐BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyve‐dependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient‐rich conditions, PI3Kα, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P2‐dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Kα signaling to lysosome reformation during basal autophagy.

Molecular characteristics of advanced colorectal cancer and multi-hit PIK3CA mutations.

3535 Background: Approximately 20% of colorectal cancer (CRC) has an activating mutation in the PIK3CA oncogene. PIK3CA codes for the catalytic subunit of phosphoinositide 3-kinase alpha (PI3Kα), which ultimately activates the AKT and mammalian target of rapamycin (mTOR) pathway. The PI3Kα inhibitor alpelisib has been approved for breast cancer where a single PIK3CA activating mutation is sufficient for response. However, two activating mutations (multi-hit) in the PIK3CA allele substantially increases PI3Kα signaling compared to single hot spot mutations and results in exceptional response to PI3Kα inhibition. We aimed to identify the prevalence of PIK3CA multi-hit mutations in CRC to identify patients potentially susceptible to PI3K inhibitors. Methods: Tissue-based comprehensive genomic profiling (CGP) was performed in a Clinical Laboratory Improvement Amendments (CLIA)-certified, CAP (College of American Pathologists)-accredited laboratory (Foundation Medicine Inc., Cambridge, MA, USA) on all-comers during the course of routine clinical care from 2013-2021. Approval was obtained from the Western Institutional Review Board (Protocol No. 20152817). Hybrid capture was carried out for at least 324 cancer-related genes, including PIK3CA. Results: We identified 48,836 patients with advanced CRC who underwent Foundation Medicine testing and 846 (1.7%) patients with multi-hit PIK3CA mutations. Additional clinical and molecular data was available for 41,154 of these patients of which 710 (1.7%) had multi-hit PIK3CA and 7627 (19%) had any deleterious PIK3CA mutation . The local colon tumor was used for sequencing in 70% of cases and a separate site in 30% of cases. Patients with PIK3CA multi-hit mutations were 53% male with a median age of 60 (interquartile range 50-70). The microsatellite status was available for 697 of 710 patients with multi-hit PIK3CA and 123/697 (18%) were microsatellite instability-high. The Table outlines the genes with cooccurring mutations of >10% prevalence for multi-hit PIK3CA CRC, including the clinically relevant mutations in KRAS (65%) and BRAF (13%). The four most common PIK3CA variants were H1047R (9.8%), E545K (9.2%), E542K (9.0%) and R88Q (7.1%). The most common variant pair was E542K with E545K in 4.7% of multi-hit cases. Conclusions: Double-hit mutations in PIK3CA are seen in 1.7% of advanced CRC patients and may represent a subset of patients that may have enhanced sensitivity to PI3K inhibitors. Given the high prevalence of CRC in the United States and worldwide this represents a clinically meaningful prevalence of multi-hit PIK3CA. Future investigation on the clinical utility of PI3K inhibitors may be warranted in multi-hit PIK3CA CRC.[Table: see text]

PI 3-kinase isoform PI3Kalpha controls smooth muscle cell functionality and protects against aortic aneurysm formation

Abstract Background Class I PI 3-kinase isoform PI3Kα is a lipid kinase and signals downstream of receptor tyrosine kinases. Smooth muscle cells (SMCs) lacking PI3Kα are characterized by impaired proliferation, migration and survival. Mice, harbouring a smooth muscle specific PI3Kα deficiency (SM-PI3Kα−/−), display reduced vascular wall thickness and impaired vascular remodeling in response to vessel injury. We hypothesize that SM-PI3Kα−/− mice are prone to aortic aneurysm (AA) formation due to impaired SMC functions. Herein, we investigated, how PI3Kα-dependent signaling in SMCs affect aortic aneurysm (AA) formation, aortic wall structure, and expression of extracellular matrix (ECM) components. Methods and results AA formation in SM-PI3Kα−/− mice and wild-type littermates was examined by means of the “porcine pancreatic elastase” (PPE) AA model. PPE was infused into the infrarenal aorta to induce AA formation. Ultrasound examination revealed a significantly increased aortic diameter in SM-PI3Kα−/− mice (1.22±0.12 mm) compared to wild-type animals (0.96±0.02 mm, p=0.014). These data indicate a protective function of SM-PI3Kα in AA formation. In addition, the media thickness in the abdominal aorta was significantly reduced in SM-PI3Kα−/− mice (29.0±3.1 vs. 42.5±4.1 μm). Ultrastructural analysis of aortic wall morphology in SM-PI3Kα−/−mice using transmission electron microscopy (TEM) showed a deranged tunica media with detached SMCs and increased apoptotic cell death. Consequently, SM-PI3Kα deficiency significantly diminished responsiveness of aortic rings to vasodilator acetylcholine and NO-donor nitroglycerin, further indicating impaired aortic wall structure. Western blots demonstrated a reduced elastin and fibrillin expression in SMCs from SM-PI3Kα−/− mice. Furthermore, immunofluorescence stainings of PI3Kα−/− and wild-type SMCs, cultured for seven days under 10% fetal calf serum containing DMEM medium, showed significantly disturbed structures of elastin-, fibrillin-1- and collagen-1-fibers. These data indicate that PI3Kα signaling contributes to elastic fiber homeostasis thus affecting SMC phenotypic modulation. Immunoblots demonstrated that PDGF and insulin induced phosphorylation and inactivation of key regulators of SMC differentiation and dedifferentiation including FoxO1, FoxO3a, Foxo4, and GSK3b, respectively, were reduced or even abrogated in PI3Kα−/− SMCs. Conclusion These data show that deficiency of PI3Kα in SMCs promotes the formation and progression of AA. Causative is a deranged aortic structure of SM-PI3Kα−/− aortae which can likely be attributed to an impaired production of elastic fiber components by PI3Kα−/− SMCs. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Abstract 1378: A novel, highly potent PI3Kα covalent inhibitor deconvolutes class I PI3K isoforms in cancer cells

Abstract Phosphoinositide 3-kinase (PI3K) imposes central regulatory signals that promote cell growth, proliferation, motility, and suppression of apoptosis. PI3K is dysregulated in a broad spectrum of human tumours.[1] Considering the central role attributed to PI3K signaling in cancer biology, the success of clinical trials in the field was below expectations, mainly due to metabolic on-target adverse effects. Here, we present a novel, highly selective PI3Kα-covalent inhibitor (compound 19), which combines the exceptional affinity of the pan-PI3K inhibitor PQR514[2] with a linker moiety, directing a warhead to selectively react via a Michael addition with isoform-specific Cys862 of PI3Kα. Cellular assay platforms were combined to validate 19, specifically, to assess its target selectivity and occupancy, and to translate its in vitro activity to cellular readouts. Overall, 19 exhibited excellent cellular activity with a superior profile compared to CNX-1351[3], a previously reported covalent PI3Kα inhibitor. A NanoBRET (Bioluminescence Resonance Energy Transfer) drug-target engagement assay allowed for an investigation of inhibitory efficacy in HEK293 cells. NanoBRET experiments exploiting the Cys862Ser mutant PI3Kα protein confirmed selective reactivity with Cys862. Various cell lines either constitutively activated in PI3Kα (MCF7, T47D, SKOV3) or deficient in PTEN (PC3 and A2058) were used to resolve target engagement, which confirmed a persistent and prolonged suppression of protein kinase B (PKB/Akt, Ser473) phosphorylation after washout of 19 over non-covalent structural analogs thereof that lack a functional warhead. Furthermore, nucleo-cytoplasmic translocation probes based on the FOXO1 transcription factor enabled the investigation of signal propagation downstream of PKB/Akt. In PI3Kα-dependent cell lines, FOXO1, maintained a predominant nuclear localization even after removal of 19, confirming extended PI3Kα targeting upon covalent attachment of specifically this drug. Importantly, 19 provided a considerable gain in potency over BYL719, a clinically approved non-covalent PI3Kα-selective inhibitor. Taken together, our results present a major step towards an increased local and temporal control of PI3Kα signaling through isoform-selective covalent anchoring. This strategy is a step towards the development of drug-like, isoform-selective, covalent PI3K inhibitor scaffolds, and 19 provides a template for the development of PI3Kα inhibitors with overall reduced adverse side effects in cancer therapy. Moreover, our covalent inhibition strategy will pave the way for a better molecular understanding of PI3K isoform signaling in general. [1] Wymann M. P. and Schneiter R. Nat. Rev. Mol. Cell Biol. 2008, 9, 162-176. [2] Borsari, C et. al. ACS Med. Chem. Lett. 2019, 10 (10), 1473-1479. [3] Nacht M et. al. J. Med. Chem. 2013, 56 (3), 712-721. Citation Format: Erhan Keles, Chiara Borsari, Rohitha Sriramaratnam, Thorsten Schäfer, Matthias P. Wymann. A novel, highly potent PI3Kα covalent inhibitor deconvolutes class I PI3K isoforms in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1378.

Loss of Phosphatidylinositol 3-Kinase Activity in Regulatory T Cells Leads to Neuronal Inflammation

Class I PI3K enzymes are critical for the maintenance of effective immunity. In T cells, PI3Kα and PI3Kδ are activated by the TCR and costimulatory receptors, whereas PI3Kγ is activated by G protein-coupled chemokine receptors. PI3Kδ is a key regulator of regulatory T (Treg) cell function. PI3K isoform-selective inhibitors are in development for the treatment of diseases associated with immune dysregulation, including chronic inflammatory conditions, cancer, and autoimmune diseases. Idelalisib (PI3Kδ), alpelisib (PI3Kα), duvelisib (PI3Kδ/γ), and copanlisib (pan-PI3K) have recently been approved for use in cancer treatment. Although effective, these therapies often have severe side effects associated with immune dysregulation and, in particular, loss of Treg cells. Therefore, it is important to gain a better understanding of the relative contribution of different PI3K isoforms under homeostatic and inflammatory conditions. Experimental autoimmune encephalitis is a mouse model of T cell-driven CNS inflammation, in which Treg cells play a key protective role. In this study, we show that PI3Kδ is required to maintain normal Treg cell development and phenotype under homeostatic conditions but that loss of PI3Kδ alone in Treg cells does not lead to autoimmunity. However, combined loss of PI3Kα and PI3Kδ signaling resulted in increased experimental autoimmune encephalitis disease severity. Moreover, mice lacking PI3Kα and PI3Kδ in Treg cells developed spontaneous peripheral nerve inflammation. These results show a key role for PI3K signaling in Treg cell-mediated protection against CNS inflammation.

Test measuring signaling activity in live patient tumor cells to identify PI3KCA WT patients who may benefit from PI3K inhibitors.

e13000 Background: Biological factors other than PIK3CA status, such as aberrant GPCR-linked signaling, may be important to measure when identifying patients eligible for PI3K inhibitors. A new assay, the CELx PI3K test, using an impedance biosensor was developed to measure ex vivo live tumor cell response to specific S1P agonists and PI3K antagonists to diagnose breast tumors with PI3K-involved hyperactive signaling. This study set out to: 1) compare CELx PI3K test results and xenograft results using cell lines with PIK3CA mutations; and 2) assess whether PI3K-involved hyperactive S1P signaling is found in PIK3CA WT breast cancer patient tumors. Methods: A panel of 17 fresh HER2-/PIK3CA WT tumor cells from breast cancer patients and three PIK3CA mutated breast tumor cell lines were obtained. Live cell response to an S1P agonist, PI3K-α antagonist (alpelisib), PI3K-γ antagonist (IPI-549), and a pan-PI3K inhibitor (taselisib) were measured using an xCELLigence RTCA impedance biosensor. From these responses, PI3K-involved signaling was quantified and characterized as normal or abnormal using a previously determined cutpoint. For the xenograft study, 16 NSG mice were injected with HCC1954 PIK3CA mutated breast cancer cells and randomly assigned to either the control or taselisib group (10 mg/kg). Results: Four of the 17 PIK3CA WT tumor cells had abnormal levels of combined PI3K-α and PI3K-γ signaling. Only one of the three PIK3CA mutated breast tumor cell lines (BT20) had abnormal levels of PI3K-α and pan-PI3K involved signaling. The HCC1954 cell line had normal PI3K-α and abnormal pan-PI3K signaling. CAL-51 reported normal PI3K-α and pan-PI3K signaling. The normal levels of PI3K-α signaling found in the HCC1954 and CAL-51 cell lines correlated with previously reported xenograft studies that found alpelisib had no anti-tumor effect. The xenograft study reported here using HCC1954 cells found taselisib induces a significant anti-tumor effect (T/C ratio = 0.21; p = 0.009; t-test). Conclusions: A sub-set of PIK3CA WT patient breast cancer tumors had abnormal PI3K-involved signaling comparable to levels found in PI3KCA mutated cell lines. Abnormal pan-PI3K signaling and normal PI3K-α signaling in the HCC1954 cell line correlated with xenograft results. This study thus suggests that measurement of PI3K-involvement in hyperactive S1P signaling in live patient breast cancer cells may provide a means to identify breast cancer patients who may or may not benefit from treatment with PI3K inhibitors.

Cobimetinib plus gemcitabine is an active combination in KRAS G12R-mutated in previously chemotherapy-treated and failed pancreatic patients.

4642 Background: he KRAS proto-oncogene is involved in the RAS/MAPK pathway. Various G12X mutations have been examined with the most common mutations being G12D (40%), G12V (30%), and G12R (15-20%) in pancreatic cancer patients. Throughout the course of studying the G12X mutations, we have observed that not all KRAS mutations are equal. Preclinical data shows G12R is impaired in pI3Kα signaling, as compared to KRAS G12V/D. This mechanism is important in PDAC as it allows tumor growth to be sustained. In preclinical studies, PDX derived tumors were transplanted in mice and were treated with a MEK inhibitor plus chemotherapy, which demonstrated a greater tumor regression than either agent alone. Therefore, we have decided to treat patients with Gemcitabine alongside a 2nd generation MEK inhibitor (Cobimetinib). Methods: In our single arm study, 13 KRAS mutated pancreatic patients (KRAS G12D, G12V, and G12R) received the combination of Cobimetinib 20mg BID weekly for three weeks alongside Gemcitabine at 1000mg/m2 weekly, followed by one week of rest. The above constitutes one cycle. Results: Patients were divided into two groups; Group 1 consists of seven patients that were KRAS G12D/G12V mutated, and Group 2 included six KRAS G12R mutated patients. In Group 1, seven patients on treatment progressed and died within two months on the study. In Group 2, one achieved PR and others stable disease. Median progression-free survival was 6.0 months (95% CI 3-9.3 months) and median OS has not been reached. All patients are alive at 8 months. Common adverse reactions include rash, fatigue, nausea, and vomiting. Cancer antigen 19-9 decreased in ≥ 50 of all patients in the latter group. We would like to report our positive study to the society. Moreover, we intend to confirm the study in a larger patient cohort. Conclusions: Pancreatic cancer patients that demonstrate KRAS G12R mutations are treatable with a new active combination chemotherapy.

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.

PI3Kα Pathway Inhibition With Doxorubicin Treatment Results in Distinct Biventricular Atrophy and Remodeling With Right Ventricular Dysfunction.

BackgroundCancer therapies inhibiting PI3Kα (phosphoinositide 3‐kinase‐α)–dependent growth factor signaling, including trastuzumab inhibition of HER2 (Human Epidermal Growth Factor Receptor 2), can cause adverse effects on the heart. Direct inhibition of PI3Kα is now in clinical trials, but the effects of PI3Kα pathway inhibition on heart atrophy, remodeling, and function in the context of cancer therapy are not well understood.Method and ResultsPharmacological PI3Kα inhibition and heart‐specific genetic deletion of p110α, the catalytic subunit of PI3Kα, was characterized in conjunction with anthracycline (doxorubicin) treatment in female murine models. Biventricular changes in heart morphological characteristics and function were analyzed, with molecular characterization of signaling pathways. Both PI3Kα inhibition and anthracycline therapy promoted heart atrophy and a combined effect of distinct right ventricular dilation, dysfunction, and cardiomyocyte remodeling in the absence of pulmonary arterial hypertension. Congruent findings of right ventricular dilation and dysfunction were seen with pharmacological and genetic suppression of PI3Kα signaling when combined with doxorubicin treatment. Increased p38 mitogen‐activated protein kinase activation was mechanistically linked to heart atrophy and correlated with right ventricular dysfunction in explanted failing human hearts.Conclusions The PI3Kα pathway promotes heart atrophy in mice. The right ventricle is specifically at risk for dilation and dysfunction in the setting of PI3K inhibition in conjunction with chemotherapy. Inhibition of p38 mitogen‐activated protein kinase is a proposed therapeutic target to minimize this mode of cardiotoxicity.

Functional significance of co-occurring mutations in PIK3CA and MAP3K1 in breast cancer.

The PI3Kα signaling pathway is frequently hyper-activated in breast cancer (BrCa), as a result of mutations/amplifications in oncogenes (e.g. HER2), decreased function in tumor suppressors (e.g. PTEN) or activating mutations in key components of the pathway. In particular, activating mutations of PIK3CA (~45%) are frequently found in luminal A BrCa samples. Genomic studies have uncovered inactivating mutations in MAP3K1 (13-20%) and MAP2K4 (~8%), two upstream kinases of the JNK apoptotic pathway in luminal A BrCa samples. Further, simultaneous mutation of PIK3CA and MAP3K1 are found in ~11% of mutant PIK3CA tumors. How these two alterations may cooperate to elicit tumorigenesis and impact the sensitivity to PI3K and AKT inhibitors is currently unknown. Using CRISPR gene editing we have genetically disrupted MAP3K1 expression in mutant PIK3CA cell lines to specifically create in vitro models reflecting the mutational status of PIK3CA and MAP3K1 in BrCa patients. MAP3K1 deficient cell lines exhibited ~2.4-fold increased proliferation rate and decreased sensitivity to PI3Kα/δ(AZD8835) and AKT (AZD5363) inhibitors (~2.61 and ~5.23-fold IC50 increases, respectively) compared with parental control cell lines. In addition, mechanistic analysis revealed that MAP3K1 disruption enhances AKT phosphorylation and downstream signaling and reduces sensitivity to AZD5363-mediated pathway inhibition. This appears to be a consequence of deficient MAP3K1-JNK signaling increasing IRS1 stability and therefore promoting IRS1 binding to p85, resulting in enhanced PI3Kα activity. Using 3D-MCF10A-PI3KαH1047R models, we found that MAP3K1 depletion increased overall acinar volume and counteracted AZD5363-mediated reduction of acinar growth due to enhanced proliferation and reduced apoptosis. Furthermore, in vivo efficacy studies revealed that MAP3K1-deficient MCF7 tumors were less sensitive to AKT inhibitor treatment, compared with parental MCF7 tumors. Our study provides mechanistic and in vivo evidence indicating a role for MAP3K1 as a tumor suppressor gene at least in the context of PIK3CA-mutant backgrounds. Further, our work predicts that MAP3K1 mutational status may be considered as a predictive biomarker for efficacy in PI3K pathway inhibitor trials.

Oncogenic mutations weaken the interactions that stabilize the p110α-p85α heterodimer in phosphatidylinositol 3-kinase α.

Phosphatidylinositol 3-kinase (PI3K) α is a heterodimeric lipid kinase that catalyzes the conversion of phosphoinositol-4,5-bisphosphate to phosphoinositol-3,4,5-trisphosphate. The PI3Kα signaling pathway plays an important role in cell growth, proliferation, and survival. This pathway is activated in numerous cancers, where the PI3KCA gene, which encodes for the p110α PI3Kα subunit, is mutated. Its mutation often results in gain of enzymatic activity; however, the mechanism of activation by oncogenic mutations remains unknown. Here, using computational methods, we show that oncogenic mutations that are far from the catalytic site and increase the enzymatic affinity destabilize the p110α-p85α dimer. By affecting the dynamics of the protein, these mutations favor the conformations that reduce the autoinhibitory effect of the p85α nSH2 domain. For example, we determined that, in all of the mutants, the nSH2 domain shows increased positional heterogeneity as compared with the wild-type, as demonstrated by changes in the fluctuation profiles computed by normal mode analysis of coarse-grained elastic network models. Analysis of the interdomain interactions of the wild-type and mutants at the p110α-p85α interface obtained with molecular dynamics simulations suggest that all of the tumor-associated mutations effectively weaken the interactions between p110α and p85α by disrupting key stabilizing interactions. These findings have important implications for understanding how oncogenic mutations change the conformational multiplicity of PI3Kα and lead to increased enzymatic activity. This mechanism may apply to other enzymes and/or macromolecular complexes that play a key role in cell signaling.

Feedback Suppression of PI3Kα Signaling in PTEN-Mutated Tumors Is Relieved by Selective Inhibition of PI3Kβ

In PTEN-mutated tumors, we show that PI3Kα activity is suppressed and PI3K signaling is driven by PI3Kβ. A selective inhibitor of PI3Kβ inhibits the Akt/mTOR pathway in these tumors but not in those driven by receptor tyrosine kinases. However, inhibition of PI3Kβ only transiently inhibits Akt/mTOR signaling because it relieves feedback inhibition of IGF1R and other receptors and thus causes activation of PI3Kα and a rebound in downstream signaling. This rebound is suppressed and tumor growth inhibition enhanced with combined inhibition of PI3Kα and PI3Kβ. In PTEN-deficient models of prostate cancer, this effective inhibition of PI3K causes marked activation of androgen receptor activity. Combined inhibition of both PI3K isoforms and androgen receptor results in major tumor regressions.

Dual loss of PI3Kα and PI3Kγ signaling leads to an age-dependent cardiomyopathy

Phosphatidylinositide 3-kinase (PI3K) signaling plays a critical role in maintaining normal cardiac structure and function. PI3Kα and PI3Kγ are the dominant cardiac isoforms and have both adaptive and maladaptive roles in heart disease. Broad spectrum PI3K inhibitors are emerging as potential new chemotherapeutic agents which may have deleterious long-term effects on the heart. We created a double mutant (PI3KDM) model by crossing p110γ−/− (PI3KγKO) with cardiac-specific PI3KαDN mice and studied cardiac structure and function at 1-year of age. Pressure–volume loop analysis and echocardiographic assessment showed PI3KDM mice developed marked impairment in systolic function while the wildtype, PI3KαDN, and PI3KγKO mice maintained normal systolic and diastolic function at 1-year of age. The PI3KDM hearts displayed increased expression of disease markers, increased myocardial fibrosis and matrix metalloproteinase (MMP) activity, depolymerization of intracellular F-actin, loss of phospho(threonine-308)-Akt, and normalization of phospho-Erk1/2 signaling. Dual loss of PI3Kα and PI3Kγ isoforms results in an age-dependent cardiomyopathy implying that long-term exposure to pan-PI3K inhibitors may lead to severe cardiotoxicity.

Characterization of the Novel and Specific PI3Kα Inhibitor NVP-BYL719 and Development of the Patient Stratification Strategy for Clinical Trials

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Kα may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Kα and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose- and time-dependent inhibition of PI3Kα signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.

Abstract C170: GDC-0941 inhibits cytarabine-induced PI3K/AKT signaling and promotes synergistic activity in acute myeloid leukemia.

Abstract Introduction: We previously identified genes associated with cytarabine (AraC) response in acute myeloid leukemia (AML) and demonstrated increased expression of PIK3C3 to be significantly associated with a detrimental pattern of association (p=0.016) and multiple phenotypes (increased DNA synthesis, increased blast AraC IC50, poor clinical response, and worse event free survival) (Lamba et al., 2011). Here we evaluate the anti-leukemic activity of a selective PI3K inhibitor, GDC-0941, alone and in combination with AraC. Methods and Materials: A panel of 6 AML cell lines were assessed for potential activity of GDC-0941 alone (0.0625-4µM) and in combination with AraC (72h continuous, 4h pulse sequential or simultaneous exposure). We assessed cell viability by MTT assay and implemented the median effect approach to determine if combination drug effects, assessed as the combination index (CI) value, were synergistic, additive, or antagonistic. Induction of apoptosis and alterations in cell cycle were detected by caspase activation and propidium iodide staining, respectively. Western blot analyses were performed to determine drug effects on expression of PI3K isoforms, pAkt, pS6, and pErk signaling. Ex vivo cytotoxicity assays were performed on leukemic blasts isolated from murine primary c-Myc recipients to determine sensitivity to GDC-0941, AraC, and the combination. To evaluate in vivo efficacy, survival studies were performed using a c-Myc induced murine model of AML. Sublethally irradiated (550cGy) syngeneic tertiary recipients were randomized to receive vehicle, GDC-0941 (50mg/kg daily x 5 days, po), AraC (100mg/kg daily x 5 days, ip) or combination (week 1: AraC100mg/kg daily x 5 days; week 2: GDC-0941 50mg/kg daily x 5,) (N=8/treatment arm). Pharmacodynamic assessment of pAkt levels in leukemic blasts among the different treatment arms were also evaluated. Results: GDC-0941 treatment reduced cell viability in a dose-dependent manner and IC50s ranged from 0.36-4µM in AML cell lines. Caspase activity was induced by GDC-0941 alone and enhanced with the combination. For combination treatment, CI values were synergistic in all but 1 cell line (range, 0.20-0.97). Western blot analyses of the U937 cell line revealed that AraC induced both PI3Kα and pAkt signaling which could be abrogated by cotreatment with GDC-0941. Ex vivo combination drug treatments of c-Myc leukemic blasts demonstrated synergistic CI values. Combination treatment significantly prolonged survival in the c-Myc AML model compared to either drug alone or controls (control, median survival=21; GDC-0941, median survival=22; AraC, median survival=26; combination, median survival=28 days; p=0.01 and p=0.03 for AraC vs control and combination vs AraC, respectively) Conclusions: Our results show that AraC mediated induction of PI3Kα and pAkt in AML cells can be augmented by selective targeting of the PI3K/Akt pathway with GDC-0941. From these lines of preclinical evidence, combination of AraC with PI3K inhibitors is a promising strategy to enhance AraC efficacy in AML. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C170. Citation Format: Christina D. Drenberg, Brian Fermanski, Steven Zatechka, Yiping Fan, Shelley Orwick, Laura Janke, Sharyn D. Baker. GDC-0941 inhibits cytarabine-induced PI3K/AKT signaling and promotes synergistic activity in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C170.

Exercise training and PI3Kα-induced electrical remodeling is independent of cellular hypertrophy and Akt signaling

In contrast with pathological hypertrophy, exercise-induced physiological hypertrophy is not associated with electrical abnormalities or increased arrhythmia risk. Recent studies have shown that increased cardiac-specific expression of phosphoinositide-3-kinase-α (PI3Kα), the key mediator of physiological hypertrophy, results in transcriptional upregulation of ion channel subunits in parallel with the increase in myocyte size (cellular hypertrophy) and the maintenance of myocardial excitability. The experiments here were undertaken to test the hypothesis that Akt1, which underlies PI3Kα-induced cellular hypertrophy, mediates the effects of augmented PI3Kα signaling on the transcriptional regulation of cardiac ion channels. In contrast to wild-type animals, chronic exercise (swim) training of mice (Akt1−/−) lacking Akt1 did not result in ventricular myocyte hypertrophy. Ventricular K+ current amplitudes and the expression of K+ channel subunits, however, were increased markedly in Akt1−/− animals with exercise training. Expression of the transcripts encoding inward (Na+ and Ca2+) channel subunits were also increased in Akt1−/− ventricles following swim training. Additional experiments in a transgenic mouse model of inducible cardiac-specific expression of constitutively active PI3Kα (icaPI3Kα) revealed that short-term activation of PI3Kα signaling in the myocardium also led to the transcriptional upregulation of ion channel subunits. Inhibition of cardiac Akt activation with triciribine in this (inducible caPI3Kα expression) model did not prevent the upregulation of myocardial ion channel subunits. These combined observations demonstrate that chronic exercise training and enhanced PI3Kα expression/activity result in transcriptional upregulation of myocardial ion channel subunits independent of cellular hypertrophy and Akt signaling.