Phosphoinositide 3-kinase Alpha Research Articles

A deep learning-based theoretical protocol to identify potentially isoform-selective PI3Kα inhibitors.

Phosphoinositide 3-kinase alpha (PI3Kα) is one of the most frequently dysregulated kinases known for their pivotal role in many oncogenic diseases. While the side effects linked to existing drugs against PI3Kα-induced cancers provide an avenue for further research, the significant structural conservation among PI3Ks makes it extremely difficult to develop new isoform-selective PI3Kα inhibitors. Embracing this challenge, we herein designed a hybrid protocol by integrating machine learning (ML) with in silico drug-designing strategies. A deep learning classification model was developed and trained on the physicochemical descriptors data of known PI3Kα inhibitors and used as a screening filter for a database of small molecules. This approach led us to the prediction of 662 compounds showcasing appropriate features to be considered as PI3Kα inhibitors. Subsequently, a multiphase molecular docking was applied to further characterize the predicted hits in terms of their binding affinities and binding modes in the targeted cavity of the PI3Kα. As a result, a total of 12 compounds were identified whereas the best poses highlighted the efficiency of these ligands in maintaining interactions with the crucial residues of the protein to be targeted for the inhibition of associated activity. Notably, potential activity of compound 12 in counteracting PI3Kα function was found in a previous in vitro study. Following the drug-likeness and pharmacokinetic characterizations, six compounds (compounds 1, 2, 3, 6, 7, and 11) with suitable ADME-T profiles and promising bioavailability were selected. The mechanistic studies in dynamic mode further endorsed the potential of identified hits in blocking the ATP-binding site of the receptor with higher binding affinities than the native inhibitor, alpelisib (BYL-719), particularly the compounds 1, 2, and 11. These outcomes support the reliability of the developed classification model and the devised computational strategy for identifying new isoform-selective drug candidates for PI3Kα inhibition.

Structural insights into the activation mechanism of phosphoinositide 3-kinase alpha

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases known to regulate important cellular functions by phosphorylating the inositol ring of inositol-phospholipids (PtdIns) at 3’ position. The PI3Kα is a heterodimer and the activation of the catalytic subunit (p110α) is regulated by its regulatory subunit (p85α). The current work deals with studying the activation mechanism of the PI3Kα using multi micro-second molecular dynamic simulations. Structural changes involved in activation mechanism is studied by gradually releasing the inhibitory effects of different domains of regulatory subunit namely, n-terminal SH2 (nSH2) and inter SH2 (iSH2). The observation shows that even in the presence of n-terminal and inter SH2 domain (niSH2) of regulatory subunit, the catalytic domain has some intrinsic activation activity and the presence of c-terminal SH2 (cSH2) domain may be required for complete inhibition. The release of nSH2 domain leads to loss of interactions between iSH2 domain (regulatory subunit) and C2 and kinase domain (catalytic subunit). The study shows that early events in the activation mechanism involve the movement of the ABD domain of the catalytic subunit along with the linker region between ABD and RBD region which may lead to movement of ABD closer to the CLobe of the kinase domain. This movement is essentially as it triggers the rearrangement of CLobe especially the catalytic loop and activation loop which bring catalytic important residues closer to ATP and PIP2(phosphatidylinositol-4,5-bisphosphate). Water mediated interaction analysis reveal that water may be playing an important role in the transfer of phosphate from ATP to PIP2. The study shows that initial signal for release of inhibitory effect of the regulatory subunit might be propagated through the linker region between ABD and RBD through allosteric effect to different regions of the protein. These understanding of early events during the activation mechanism may help in the design of better therapeutic targeting PI3K.

Structural insights into the interaction of three Y-shaped ligands with PI3Kα

Class IA phosphoinositide 3-kinase alpha (PI3Kα) is an important drug target because it is one of the most frequently mutated proteins in human cancers. However, small molecule inhibitors currently on the market or under development have safety concerns due to a lack of selectivity. Therefore, other chemical scaffolds or unique mechanisms of catalytic kinase inhibition are needed. Here, we report the cryo-electron microscopy structures of wild-type PI3Kα, the dimer of p110α and p85α, in complex with three Y-shaped ligands [cpd16 (compound 16), cpd17 (compound 17), and cpd18 (compound 18)] of different affinities and no inhibitory effect on the kinase activity. Unlike ATP-competitive inhibitors, cpd17 adopts a Y-shaped conformation with one arm inserted into a binding pocket formed by R770 and W780 and the other arm lodged in the ATP-binding pocket at an angle that is different from that of the ATP phosphate tail. Such a special interaction induces a conformation of PI3Kα resembling that of the unliganded protein. These observations were confirmed with two isomers (cpd16 and cpd18). Further analysis of these Y-shaped ligands revealed the structural basis of differential binding affinities caused by stereo- or regiochemical modifications. Our results may offer a different direction toward the design of therapeutic agents against PI3Kα.

Abstract OT3-08-01: A phase 1 trial of LOXO-783, a potent, highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor in PIK3CA H1047R-mutant advanced breast cancer (aBC) and other solid tumors (PIKASSO-01, trial in progress)

Abstract Background: Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of breast cancers (BC). Available PI3Kα inhibitors target both wild-type (WT) and mutant PI3Kα and, as a result, their efficacy may be limited by on-target WT PI3Kα-mediated toxicities, including hyperglycemia, skin rash, and diarrhea. LOXO-783 is an oral, potent and highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor. Preclinically, LOXO-783 is highly selective for PI3Kα H1047R over WT PI3Kα and other PI3K isoforms and induces single-agent tumor regressions in ER+, HER2- PI3Kα H1047R-mutant BC models without causing hyperglycemia or increases in plasma insulin/C-peptide. LOXO-783 also demonstrates brain penetration in vivo with dose-dependent tumor growth inhibition in brain metastasis models. This trial investigates LOXO-783 alone and in combination with other anticancer therapies in patients with PIK3CA H1047R-mutant aBC and other solid tumors. Trial Design: This global, first-in-human phase 1a/b study of LOXO-783 includes dose escalation of LOXO-783 monotherapy followed by dose expansion of LOXO-783 alone and in combination with other anticancer therapies (Table). Monotherapy dose escalation will be evaluated using a modified toxicity probability interval-2 (mTPI-2) design. In dose expansion (Parts A-E), each combination cohort will include a safety lead-in of 3-6 patients. Men and premenopausal women with ER+ aBC must receive concomitant treatment with a GnRH agonist. An optional pharmacodynamic (PD) biomarker sub-study will be conducted at select dose levels during dose escalation in patients with ER+, HER2- aBC with soft tissue disease amenable to safe repeat tumor biopsies. Eligibility criteria: Eligible patients must have PIK3CA H1047R-mutant aBC or other solid tumors with measurable disease or non-measurable bone-only disease (aBC patients only). In dose escalation, patients may have received up to 5 prior regimens. In dose expansion, prior therapy requirements are outlined in the Table below. Key exclusion criteria include prior inhibitor(s) of PI3K/AKT/mTOR (except in dose escalation or in select patients with prior intolerance of these inhibitors), colorectal cancer, endometrial cancer with concurrent PI3K/AKT/mTOR and/or RAS/RAF alterations and diabetes mellitus (DM) requiring medication (except in Part C). Study objectives: Recommended phase 2 dose (RP2D) determination; safety and tolerability assessment, PK and PD evaluation, objective response rate and clinical benefit rate assessment per RECIST v1.1. Recruitment is ongoing (PIKASSO-01, NCT05307705). Table. Dose Expansion (Phase 1b) Citation Format: Dejan Juric, Komal Jhaveri, Muralidhar Beeram, Erika Hamilton, Vincent Chau, Matthew P. Hanley, Shiyao Liu, Lillian M. Smyth, Funda Meric-Bernstam. A phase 1 trial of LOXO-783, a potent, highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor in PIK3CA H1047R-mutant advanced breast cancer (aBC) and other solid tumors (PIKASSO-01, trial in progress) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-08-01.

Abstract P4-08-02: LOXO-783: A potent, highly mutant selective and brain-penetrant allosteric PI3Kα H1047R inhibitor in combination with standard of care (SOC) treatments in preclinical PI3Kα H1047R-mutant breast cancer models

Abstract Background Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of breast cancers (BC). Early generation PI3Kα inhibitors target both wild-type (WT) and mutant PI3Kα and, as a result, their efficacy may be limited by on-target WT PI3Kα-mediated toxicities, including hyperglycemia, skin rash, and diarrhea. LOXO-783 is an oral, potent and highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor that is currently in phase 1 testing. Preclinically, LOXO-783 as a single agent is highly selective for PI3Kα H1047R over WT PI3Kα and other PI3K isoforms, and induces single-agent tumor regressions in ER+, HER2- PI3Kα H1047R-mutant breast cancer models without causing hyperglycemia or increases in plasma insulin/C-peptide. LOXO-783 also demonstrates brain penetration in vivo with dose-dependent tumor growth inhibition in brain metastasis models. Here we report the efficacy of LOXO-783 with SOC treatments in preclinical breast cancer models. Methods Cell proliferation assays and in vivo studies to evaluate combination effects were performed in various PI3Ka H1047R mutant HR+, HER2- and triple negative breast cancer models. For each combination in vitro, a combination index (CI) based on the Loewe Additivity Method was calculated (CI>2 antagonism, 0.5>CI< 2 additivity, CI< 0.5 synergy). For the in vivo studies, the Bliss Independence Method was used to evaluate the statistical significance of the combination effects. Results Combining LOXO-783 with either fulvestrant (FUL; CI at 50% inhibition = 0.28) or imlunestrant (CI at 50% inhibition = 0.43) showed increased efficacy in cell proliferation assays using the HR+, HER2-, PI3Kα H1047R-mutant T47D model. LOXO-783 also demonstrated an additive effect in combination with these endocrine therapies in vivo. Similar results were observed in a T47D model engineered to express ESR1 D538G, as well as in an HR+, HER2- PI3Kα double in-cis mutant model (H1047R/D350G) also harboring ESR1 D538G and derived from a patient who had progressed on prior letrozole plus taselisib. Moreover, LOXO-783 plus abemaciclib demonstrated an additive effect in vitro (CI at 50% inhibition = 0.61), and in T47D xenograft and PDX models in vivo. Combinations of LOXO-783 with abemaciclib plus imlunestrant resulted in a mean tumor regression of –48.1%; LOXO-783 with abemaciclib plus FUL showed mean tumor regression of –43.9% in T47D xenografts. Similar efficacy was not observed in the absence of LOXO-783 (mean tumor regression was –7.3% with abemaciclib plus imlunestrant, and 3.2% with abemaciclib plus FUL). We observed comparable results in PDX models. These data collectively demonstrate the additive effect of LOXO-783 with SOC treatments. Extending these studies to additional treatment settings, LOXO-783 was similarly efficacious as a single agent in abemaciclib-resistant and abemaciclib/FUL double-resistant models, and was additive in combination with paclitaxel in a triple negative breast cancer model in vitro and in vivo. Conclusions LOXO-783 shows additive effects when combined with SOC in breast cancers harboring the PI3Kα H1047R-mutation (as single or double in-cis mutations) in both HR+ and triple negative settings. LOXO-783 is also efficacious in ESR1 mutant as well as in abemaciclib and abemaciclib/FUL double-resistant models. A phase 1 trial of LOXO-783 alone or in combination with anticancer therapies is ongoing (PIKASSO-01; NCT05307705). Citation Format: Loredana Puca, Michele S. Dowless, Carmen M. Perez-Ferreiro, Maria Jesus Ortiz-Ruiz, Gregory P. Donoho, Andrew Capen, Lysiane Huber, Sarah M. Bogner, Dongling Fei, Jason R. Manro, Chun Ping Yu, Wei Guo Xu, Rui Wang, Shuang Chen, Mark A. Hicks, Parisa Zolfaghari, Andrew Faber, Raymond Gilmour, Monica D. Ramstetter, Matthew T. Chang, Maria Jose Lallena, Xuequian Gong, David M. Hyman, Lillian M. Smyth, Barbara J. Brandhuber, Barry S. Taylor, Anke Klippel. LOXO-783: A potent, highly mutant selective and brain-penetrant allosteric PI3Kα H1047R inhibitor in combination with standard of care (SOC) treatments in preclinical PI3Kα H1047R-mutant breast cancer models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-02.

107 (PB097) - Preclinical characterization of TOS-358, a potent and selective covalent inhibitor of wild-type and mutant PI3Kα with superior anticancer activity

Phosphoinositide 3-kinase alpha (PI3Kα) is the most frequently mutated oncogene in cancer, inferring a critical role for this protein in neoplasia. The molecular biology of PI3Kα reveals it to be a protein that integrates a large and diverse set of cellular signals. In the development of small molecule inhibitors of this target protein it has been demonstrated that deep but also durable inhibition is critical to potent anti-cancer activity, an area traditionally challenging for reversible competitive and allosteric inhibitors. TOS-358 was developed to inhibit covalently both wild-type and mutant PI3Kα with observed IC50 s of 2.2 nM for WT PI3Kα and 4.1 nM for mutant PI3Ka (H1047R). TOS-358 is highly selective in a kinome-wide screen and selective for PI3Kα over other isoforms. We confirmed covalent binding to PI3Kα by NanoBRET in washout experiments, where it was observed that TOS-358 maintained 90% binding at 100 nM 6 hours after washout; in contrast, binding of Alpelisib at 100 nM was completely lost over the same time frame. Irreversible binding was further established using TR-FRET assay in which Kinact/KI was found to be 5.6 × 107 M−1s−1. Importantly, we also noted that TOS-358 produced sustained inhibition of phosphorylated AKT(S473) to 48 hours while allosteric inhibitors lose >60% inhibition. TOS-358 mediated cell growth inhibition has been evaluated in a panel of 120 cell lines and compared with Alpelisib in the same panel. This analysis revealed approximately 50% more cell lines to be responsive to TOS-358 compared with Alpelisib. There was no strong association of response and PI3Kα mutation status, and in fact for both TOS-358 and Alpelisib there was a higher frequency of WT PI3Kα cell lines responding to either treatment. TOS-358 activity has been tested in multiple different cell-derived and patient-derived xenograft cancer models and was found to produce reproducible and substantial tumor growth inhibition. Indeed, in several PDX models, TOS-358 induced tumor regressions while the clinical stage molecules Alpelisib and Inavolisib were unable to generate similar tumor regressions despite pharmacokinetic exposures comparable to, or in excess of, TOS-358. Finally, TOS-358 generated little or no glucose impact in mice and dogs at exposures that produced superior efficacy in these cancer models. Taken together, our in vitro and in vivo data reveal TOS-358 to be a potent, selective covalent inhibitor of PI3Kα with superior anticancer activity to comparator molecules. Conflict of interest: Ownership: The authors are employees of and may hold equity in Totus Medicines.

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]

Genome-wide gain-of-function screening identifies EZH2 mediating resistance to PI3Kα inhibitors in oesophageal squamous cell carcinoma.

Phosphoinositide‐3 kinase alpha (PI3Kα) has been confirmed to be a potential therapeutic target for esophageal squamous cell carcinoma (ESCC), while the potency of PI3Kα inhibitors is often attenuated by concurrent oncogenic signalling pathways. We performed genome‐wide gain‐of‐function screening with a CRISPR‐SAM library and identified enhancer of zeste homolog 2 (EZH2) rendering ESCC cells resistant to the PI3Kα inhibitor CYH33. Enhanced expression of EZH2 frequently occurs in ESCC and is related to poor prognosis. Overexpression of full‐length EZH2 but not methyltransferase‐deficient EZH2 conferred resistance to CYH33, while downregulating EZH2 expression restored sensitivity. EZH2 expression was negatively related to the activity of CYH33 against the proliferation of ESCC cell lines and patient‐derived cells. Transcriptomic analysis revealed that EZH2 abrogated CYH33‐mediated cell cycle regulation. EZH2 epigenetically suppressed the transcription of CDKN1A, promoting RB phosphorylation and cell cycle progression. Concurrently targeting EZH2 significantly potentiated CYH33 to inhibit the growth of ESCC cells and patient‐derived xenografts accompanied by enhanced cell cycle arrest. Taken together, our study demonstrated that an EZH2‐p21‐RB axis remodeled cell cycle regulation and rendered resistance to PI3Kα inhibitors in ESCC. Simultaneously targeting PI3Kα and EZH2 may provide an effective strategy for ESCC therapy with high expression of EZH2.

Abstract P4-02-05: Exploiting novel models of endocrine-resistant breast cancer to identify new therapeutic targets

Abstract Introduction: While endocrine therapy is an effective, well-tolerated treatment for estrogen receptor positive breast cancer (ER+BC), a large proportion of initial responders will develop hormone therapy resistance, and relapse. A major challenge in determining the mechanisms underlying endocrine therapy resistance is our limited ability to recapitulate inter- and intra-tumour heterogeneity in vitro. In this study we developed ER+BC cells resistant to estrogen deprivation, and to palbociclib treatment, and subjected these to 2D and 3D high-throughput drug and siRNA screens, in order to elucidate the pathways underpinning the development of endocrine resistance. Methods: ER+BC cell lines modelling relapse on aromatase inhibitor therapy were derived by long-term culture in the absence of exogenous estrogen, and were termed long-term estrogen deprived (LTED). Two of these lines were then used to generate additional resistance to palbociclib, thus modelling endocrine-resistant, palbociclib-resistant disease. These lines were then subjected to screens performed in 2D and 3D. Effects of the siRNAs and drug compounds were assessed by measuring cell viability, with a robust Z score of -2 taken as a threshold of significance. Table 1 outlines the key characteristics of the cell lines, the details of the drug screens performed, and the top 3 drugs with their corresponding targets. The siRNA screens were performed in 2D and 3D with a 709 kinome library. Results: The siRNA screens highlighted PI3K-AKT-mTOR signalling as being dysregulated in multiple 3D and 2D models, while the drug screens showed that compounds targeting phosphoinositide 3-kinase alpha (PI3Kα) were significant common hits. This is consistent with the recent success of the BYLieve trial in targeting PI3Kα. A second key area of dependency was in cell cycle regulation, with CDK7 and CDK9 found to be significant hits in multiple models. The 709 kinase screens in 2D and 3D demonstrated broadly similar results (43 vs 37 median number of significant hits 2D vs 3D; Z-score <-2), while drug screening in 3D (Table 1) provided increased discrimination in identifying key vulnerabilities. CDK7 and CDK9 were selected for further study. siRNA-mediated knockdown of CDK9 had a significant effect on cell viability in endocrine-sensitive, endocrine-resistant, and palbociclib-resistant settings, whereas CDK7 knockdown did not. However, the CDK7 inhibitors THZ1 and ICEC0942, and the CDK9 inhibitors NVP-2 and AZD4573, were equally effective in endocrine-sensitive, endocrine-resistant, and palbociclib-resistant settings. Importantly, presence of a PIK3CA mutation was required for entry into the BYLieve trial, while CDK7 and CDK9 inhibitors were effective in the PIK3CA wild-type and mutant setting. Furthermore, while the presence of the ESR1 mutation is associated with poorer PFS with certain therapies, such as exemestane, the CDK7 and CDK9 inhibitors were equally effective in the ESR1 mutant and wild-type setting. Finally, combination treatment of palbociclib with CDK7 and CDK9 inhibitors showed synergism in palbociclib-resistant models. Conclusions: Targeted inhibition of CDK7 and/or CDK9 is effective in the LTED palbociclib-resistant setting, thus opening future avenues for the treatment of advanced, endocrine-resistant, palbociclib-resistant breast cancer. Table 1.Cell line characteristics and drug screen findingsCell line and key characteristics2D drug screen of 396 compounds, at 3 concentrations*3D drug screen of 70 compounds at 1 concentrationNumber of significant drug screen hits (Z score <-2). The top 3 hits and their targets are shownMCF7 LTEDWT6812PIK3CA mutantDinaciclib (CDKs 2/5/1/9)CUDC-907 (PI3Kα/HDAC)ESR1 wild typeTHZ1 (CDK7)PIK-75 HCl (PI3K)PIK-75 HCl (PI3Kα)Flavopiridol (CDKs 1/2/4/6)MCF7 LTEDY537C849PIK3CA mutantCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)Activating ESR1 mutation (Y537C)THZ1 (CDK7)BGT226 (PI3K/mTOR)PIK-75 HCl (PI3Kα)NVP-2 (CDK9)MCF7 LTEDY537C-PalboR(n/a)8As MCF7 LTEDY537CCUDC-907 (PI3Kα/HDAC)Palbociclib resistantPIK-75 HCl (PI3Kα)NVP-2 (CDK9)SUM44 LTEDY537S6717PIK3CA mutantDinaciclib (CDKs 2/5/1/9)NVP-2 (CDK9)Activating ESR1 mutation (Y537S)CUDC-907 (PI3Kα/HDAC)WYE-125132 (mTOR)E-cadherin nullBGT226 (PI3K/mTOR)CUDC-907 (PI3Kα/HDAC)HCC1428 LTED5114PIK3CA wild typeCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)ESR1 wild typeBGT226 (PI3K/mTOR)BGT226 (PI3K/mTOR)Torin-2 (mTOR)Sapanisertib (mTOR)HCC1428 LTEDPalboR(n/a)11As HCC1428 LTEDCUDC-907 (PI3Kα/HDAC)Palbociclib resistantCUDC-101 (EGFR)BGT226 (PI3K/mTOR)T47D LTED5712PIK3CA mutantCUDC-907 (PI3Kα/HDAC)CUDC-907 (PI3Kα/HDAC)Loss of ER expression in adapting to LTEDDinaciclib (CDKs 2/1/5/9)PIK-75 HCl (PI3Kα)THZ1 (CDK7)CUDC-101 (EGFR)ZR75.1 LTED29(n/a)PTEN nullDinaciclib (CDKs 2/5/1/9)Loss of ER expression in adapting to LTEDTHZ1 (CDK7)Omipalisib (PI3K/mTOR)LTED: long-term estrogen deprived*Number of significant hits at 100 nM dose are shown Citation Format: Arany Soosainathan, Joanna Nikitorowicz-Buniak, Sunil Pancholi, Marjan Iravani, John Alexander, Syed Haider, Stephen R Johnston, Mitchell Dowsett, Lesley A Martin, Clare M Isacke. Exploiting novel models of endocrine-resistant breast cancer to identify new therapeutic targets [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-05.

Abstract P5-16-10: RLY-2608: The first allosteric mutant- and isoform-selective inhibitor of PI3Kα, is efficacious as a single agent and drives regressions in combination with standard of care therapies in PIK3CA mutant breast cancer models

Abstract Inhibition of CDK4/6 combined with the estrogen receptor (ER) degrader fulvestrant significantly improves progression free survival and overall survival in advanced hormone receptor positive (HR+) breast cancer patients and is now the standard of care (SOC) in this disease. Up to 40% of HR+ breast cancers harbor PIK3CA mutations leading to activation of phosphoinositide 3-kinase alpha (PI3Kα), which has been associated with resistance to CDK4/6 inhibitors and fulvestrant. Therefore, PI3Kα inhibitor combinations with CDK4/6 inhibitors and/or fulvestrant are of high interest in HR+, PIK3CA mutant breast cancer. The therapeutic index of active site (orthosteric) inhibitors of PI3Kα has been limited by the dual issues of no clinically meaningful selectivity for mutant versus wild-type (WT) PI3Kα and off-isoform inhibitory activity. Alpelisib, the only approved orthosteric PI3Kα inhibitor, is emblematic of the class with toxicity related to inhibition of wild type PI3Kα and other PI3K isoforms resulting in sub-optimal inhibition of mutant PI3Kα, frequent discontinuation, and challenges in combining with CDK4/6 inhibitors. To overcome these limitations, we designed RLY-2608, the first allosteric, mutant, and isoform-selective inhibitor of PI3Kα. We solved the full-length cryo-EM structure of PI3Kα, performed long time-scale molecular dynamic simulations to elucidate conformational differences between WT and mutant PI3Kα, and leveraged these insights to enable the design of RLY-2608. RLY-2608 does not compete with orthosteric inhibitors for binding and associates 8x faster with mutant PI3Kα relative to WT PI3Kα. In biochemical assays, RLY-2608 inhibits kinase domain (H1047R) and helical domain (E542K, and E545K) mutant PI3Kα activity, demonstrating <10nM potency with 8-12x selectivity relative to WT. RLY-2608 is > 1000-fold selective over the β, δ, and γ PI3K isoforms in biochemical assays and demonstrates exquisite selectivity across a panel of 322 kinases, with no other kinases showing >50% inhibition. We performed in vitro combinations in two HR+ PIK3CA mutant cell lines (MCF7: E545K; T47D: H1047R) and observed synergy between RLY-2608 and fulvestrant or CDK4/6 inhibitors. In vivo, we tested combinations of RLY-2608 with fulvestrant and/or the CDK4/6 inhibitor abemaciclib in the MCF7 xenograft model. Oral administration of RLY-2608 in combination with fulvestrant led to improved efficacy compared to either agent alone in a dose-dependent manner, with regressions observed in the combination arms at all doses. Furthermore, the triple combination of RLY-2608, fulvestrant, and abemaciclib resulted in superior efficacy compared to either the RLY-2608 + fulvestrant or RLY-2608 + abemaciclib doublets, with deep regressions observed in the triple combination arm. In addition, in vivo combination efficacy with fulvestrant and CDK4/6 inhibitors (palbociclib or abemaciclib) was assessed in patient-derived xenografts harboring the PIK3CA H1047R or E545K mutation along with a second site PIK3CA minor mutation. In these studies, combination benefit was observed with doses of RLY-2608 significantly lower than the dose required for maximum efficacy as a single agent. RLY-2608 synergizes in vitro with both anti-estrogen and CDK4/6 inhibitors in cell models of HR+/PIK3CA mutant breast cancer. RLY-2608 can be combined with fulvestrant and CDK4/6 inhibitors in vivo with tumor regressions observed in both cell- and patient-derived xenograft models. The pre-clinical profile of RLY-2608 supports the clinical development of RLY-2608 both in single agent and combination clinical trials in patients with PIK3CA mutant tumors, including HR+/PIK3CA mutant breast cancer. Citation Format: Ermira Pazolli, Randy Kipp, Alessandro Boezio, Hakan Gunaydin, Amanda Iskandar, Matthew Zubrowski, Bret Williams, Kelley Shortsleeves, Alexandre Larivee, Tom McLean, Klaus Michelsen, Hongtao Zeng, Jonathan LaRochelle, Joe Manna, Lucian DiPietro, Andre Lescarbeau, Mary Mader, Bindu Bennet, Jeremy Wilbur, Qi Wang, Levi Pierce, Iain Martin, James Watters, Pascal Fortin, Donald Bergstrom. RLY-2608: The first allosteric mutant- and isoform-selective inhibitor of PI3Kα, is efficacious as a single agent and drives regressions in combination with standard of care therapies in PIK3CA mutant breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-16-10.

Sensitivity of Oncogenic KRAS-Expressing Cells to CDK9 Inhibition.

Oncogenic forms of KRAS proteins are known to be drivers of pancreatic, colorectal, and lung cancers. The goal of this study is to identify chemical leads that inhibit oncogenic KRAS signaling. We first developed an isogenic panel of mouse embryonic fibroblast (MEF) cell lines that carry wild-type RAS, oncogenic KRAS, and oncogenic BRAF. We validated these cell lines by screening against a tool compound library of 1402 annotated inhibitors in an adenosine triphosphate (ATP)-based cell viability assay. Subsequently, this MEF panel was used to conduct a high-throughput phenotypic screen in a cell viability assay with a proprietary compound library. All 126 compounds that exhibited a selective activity against mutant KRAS were selected and prioritized based on their activities in secondary assays. Finally, five chemical clusters were chosen. They had specific activity against SW620 and LS513 over Colo320 colorectal cancer cell lines. In addition, they had no effects on BRAFV600E, MEK1, extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3-kinase alpha (PI3Kα), AKT1, or mammalian target of rapamycin (mTOR) as tested in in vitro enzymatic activity assays. Biophysical assays demonstrated that these compounds did not bind directly to KRAS. We further identified the mechanism of action and showed that three of them have CDK9 inhibitory activity. In conclusion, we have developed and validated an isogenic MEF panel that was used successfully to identify RAS oncogenic or wild-type allele-specific vulnerabilities. Furthermore, we identified sensitivity of oncogenic KRAS-expressing cells to CDK9 inhibitors, which warrants future studies of treating KRAS-driven cancers with CDK9 inhibitors.

PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity.

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2β) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2β in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2β showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2β in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2β as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.

Abstract P3-03-01: Double PIK3CA mutations in cis drive oncogene addiction and enhance sensitivity to PI3K alpha inhibitors in breast cancer

Abstract Activating mutations in PIK3CA, the gene coding for the catalytic subunit (p110α) of phosphoinositide-3-kinase (PI3K), are the most frequent oncogenic alterations in estrogen receptor-positive (ER+) breast cancer and are also prevalent in other tumor types. PI3Kα inhibitors including alpelisib have recently been shown to be clinically active in ER+ PIK3CA mutant breast cancer. To characterize determinants of sensitivity to these agents, we undertook a comprehensive analysis of PIK3CA mutant cancer genomes and observed the presence of double PIK3CA mutations in 12-15% of breast cancer and other tumor types. These double PIK3CA mutations are clonal, located in cis on the same allele, and are composed of a single hotspot mutation combined with a recurrent second-site mutation. Double PIK3CA mutations in cis result in increased PI3K activity and downstream signaling together with enhanced cell proliferation and tumor growth compared to single hotspot mutations. The biochemical mechanisms underlying this increased oncogenicity include increased disruption of p110α binding to the inhibitory subunit p85α, which relieves its catalytic inhibition, and increased membrane lipid binding. Finally, these double PIK3CA mutations predict for increased sensitivity to PI3Kα inhibitors compared to single hotspot mutations (e.g. E545K or H1047R) in experimental models and in patients with ER+ PIK3CA mutant metastatic breast cancer from the SANDPIPER randomized phase III clinical trial. These findings implicate double PIK3CA mutations in cis as a novel mechanism of oncogene addiction relative to single hotspot mutations, providing a rationale to develop PI3Kα inhibitors for the therapy of double PIK3CA mutant cancers. Citation Format: Neil Vasan, Pedram Razavi, Jared L Johnson, Hong Shao, Timothy Wilson, Frauke Schimmoller, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Ting-Yu Lin, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Sarat Chandarlapaty, Raul Rabadan, Ed Reznik, Melissa L Smith, Robert Sebra, Lori Friedman, Lewis C Cantley, Maurizio Scaltriti, José Baselga. Double PIK3CA mutations in cis drive oncogene addiction and enhance sensitivity to PI3K alpha inhibitors in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-01.

Abstract 3917: Double PIK3CA mutations in cis enhance oncogene activation and sensitivity to PI3K alpha inhibitors in breast cancer

Abstract Activating mutations in PIK3CA, the gene coding for the catalytic subunit (p110α) of phosphoinositide 3-kinase (PI3K), are the most frequent oncogenic alterations in all cancers, including estrogen receptor-positive (ER+) breast cancer. There are many distinct oncogenic PIK3CA mutations including major hotspot mutations (e.g. E542K, E545K, H1047R) which are predictive of response to PI3Kα inhibitors. While responses to PI3Kα inhibitors can be variable, some patients with single hotspot mutant tumors derive a deep and prolonged clinical benefit. In a comprehensive analysis of the genomic data available in breast cancer, we observed double PIK3CA mutations and investigated their potential biological relevance and potential correlation with sensitivity to PI3Kα inhibitors. We detected double PIK3CA mutations in 10-15% of all PIK3CA-mutant cancers including breast cancer. Double PIK3CA mutations in breast cancer are defined by a pattern of co-expression of a major hotspot mutation associated with a recurrent second-site PIK3CA mutation (‘minor mutation’). We have found that these double PIK3CA mutations are compound, that is in cis on the same allele, using single molecule real time sequencing (SMRT-seq) of fresh breast tumor samples. Double compound PIK3CA mutations result in increased PI3K activity and downstream signaling compared to single hotspot mutants in nontransformed cells and in ER+ breast cancer cells. Double compound mutations also increase cell proliferation and xenograft growth compared to single hotspot mutants. Biochemical experiments using recombinant PI3K compound mutant protein complexes reveal a combination mechanism of action, with simultaneous PI3K destabilization and increased membrane binding compared to single hotspot mutants, leading to increased kinase activity. Importantly, these compound mutations predict for increased sensitivity to PI3Kα inhibitors compared to single hotspot mutants in both preclinical models and also in patients with breast cancer. Together, our data support that double compound PIK3CA mutations enhance the oncogenicity of single hotspot PIK3CA mutations and this increased dependency results in increased sensitivity to PI3Kα inhibitors compared to single hotspot mutations. We propose that double compound activating mutations are an additional mechanism of oncogenic transformation in addition to other known mechanisms such as gene amplification, point mutation, or gene translocation. We speculate that double compound mutant PIK3CA may function as a clinical biomarker of increased sensitivity to PI3K-directed targeted therapies and may improve the therapeutic window of PI3K inhibitors in ER+ breast cancer and other PIK3CA-mutant tumor histologies. Citation Format: Neil Vasan, Pedram Razavi, Jared Johnson, Hong Shao, Hardik Shah, Alesia Antoine, Erik Ladewig, Alexander Gorelick, Eneda Toska, Guotai Xu, Abiha Kazmi, Matthew T. Chang, Barry S. Taylor, Maura N. Dickler, Komal Jhaveri, Raul Rabadan, Ed Reznik, Melissa L. Smith, Robert Sebra, Lewis C. Cantley, Maurizio Scaltriti, Jose Baselga. Double PIK3CA mutations in cis enhance oncogene activation and sensitivity to PI3K alpha inhibitors in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3917.

Insights into the mechanism of the PIK3CA E545K activating mutation using MD simulations

Phosphoinositide 3-kinase alpha (PI3Kα) is involved in fundamental cellular processes including cell proliferation and differentiation and is frequently mutated in human malignancies. One of the most common mutations is E545K, which results in an amino acid substitution of opposite charge. It has been recently proposed that in this oncogenic charge-reversal mutation, the interactions between the protein catalytic and regulatory subunits are abrogated, resulting in loss of regulation and constitutive PI3Kα activity, which can lead to oncogenesis. To assess the mechanism of the PI3Kα E545K activating mutation, extensive Molecular Dynamics simulations were performed to examine conformational changes differing between the wild type (WT) and mutant proteins as they occur in microsecond simulations. In the E545K mutant PI3Kα, we observe a spontaneous detachment of the nSH2 PI3Kα domain (regulatory subunit, p85α) from the helical domain (catalytic subunit, p110α) causing significant loss of communication between the regulatory and catalytic subunits. We examine the allosteric network of the two proteins and show that a cluster of residues around the mutation is important for delivering communication signals between the catalytic and regulatory subunits. Our results demonstrate the dynamical and structural effects induced by the p110α E545K mutation in atomic level detail and indicate a possible mechanism for the loss of regulation that E545K confers on PI3Kα.

Impact of PI3Kα (Phosphoinositide 3-Kinase Alpha) Inhibition on Hemostasis and Thrombosis.

Objective- PI3Kα (phosphoinositide 3-kinase alpha) is a therapeutic target in oncology, but its role in platelets and thrombosis remains ill characterized. In this study, we have analyzed the role of PI3Kα in vitro, ex vivo, and in vivo in 2 models of arterial thrombosis. Approach and Results- Using mice selectively deficient in p110α in the megakaryocyte lineage and isoform-selective inhibitors, we confirm that PI3Kα is not mandatory but participates to thrombus growth over a collagen matrix at arterial shear rate. Our data uncover a role for PI3Kα in low-level activation of the GP (glycoprotein) VI-collagen receptor by contributing to ADP secretion and in turn full activation of PI3Kβ and Akt/PKB (protein kinase B). This effect was no longer observed at high level of GP VI agonist concentration. Our study also reveals that over a vWF (von Willebrand factor) matrix, PI3Kα regulates platelet stationary adhesion contacts under arterial flow through its involvement in the outside-in signaling of vWF-engaged αIIbβ3 integrin. In vivo, absence or inhibition of PI3Kα resulted in a modest but significant decrease in thrombus size after superficial injuries of mouse mesenteric arteries and an increased time to arterial occlusion after carotid lesion, without modification in the tail bleeding time. Considering the more discrete and nonredundant role of PI3Kα compared with PI3Kβ, selective PI3Kα inhibitors are unlikely to increase the bleeding risk at least in the absence of combination with antiplatelet drugs or thrombopenia. Conclusions- This study provides mechanistic insight into the role of PI3Kα in platelet activation and arterial thrombosis.

Abstract 2853: Imaging tumor metabolism to guide treatment of breast cancer with drugs targeted at PI3K alpha

Abstract PI3KCA (which encodes the phosphoinositide-3 kinase alpha isoform) is the most frequently mutated oncogene in breast cancer. Recently, several alpha isoform-specific PI3K inhibitors have been developed and have entered into early-phase clinical trials. However, intrinsic and acquired resistance limits their utility. Non-invasive imaging methods could be used to guide treatment by not only allowing selection of the most effective drug in individual patients, but also by detecting intrinsic and acquired resistance. We have been evaluating the effectiveness of a selective PI3K alpha inhibitor, Taselisib (GDC-0032 Genentech S.A.), by monitoring the rate of [1-13C]lactate production after injection of hyperpolarized [1-13C]pyruvate in cell lines and patient-derived xenografts (PDX) models of breast cancer using 13C magnetic resonance spectroscopic imaging. Hyperpolarization of the 13C nucleus increases its sensitivity to detection in the MR experiment by 104 - 105x, thus allowing real time imaging of tumor metabolism. Preliminary data suggest that labeled lactate production can distinguish sensitive and resistant breast cancer models to Taselisib a few days after treatment. We are currently broadening the panel of PDXs analyzed and also including genetically engineered mechanistic-models of drug resistance. The present study highlights the effectiveness of hyperpolarized 13C spectroscopy/spectroscopic imaging for detecting early response or resistance to next generation PI3K inhibitors. This clinically applicable technique can provide insights into in vivo response in real-time and identify new strategies to increase the efficacy of therapy in individual patients. Citation Format: Susana Ros, Paula D'Santos, De-en Hu, Ankita S. Batra, Alan J. Wright, Elizabeth Mannion, Alejandra Bruna, Carlos Caldas, Kevin M. Brindle. Imaging tumor metabolism to guide treatment of breast cancer with drugs targeted at PI3K alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2853. doi:10.1158/1538-7445.AM2017-2853

Abstract 370: The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action

Abstract Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha sparing inhibition of p110beta, is investigated in these preclinical studies. Taselisib demonstrates greater potency in cancer cell lines harboring activating mutations in PIK3CA vs. wild-type lines, and induces regressions at tolerated doses in both PIK3CA mutant xenograft and patient-derived xenograft (PDX) models. When comparing taselisib to other clinical-stage PI3K inhibitors at Maximum Tolerated Dose (MTD) in vivo, taselisib confers greater activity in PIK3CA mutant models, which may indicate a larger therapeutic index. Unlike other PI3K inhibitors, taselisib has a gain of potency in PI3K alpha mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PI3K alpha mutant cells. Other clinical PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not have improved potency in PI3K alpha mutant cells due to their inability to maintain pathway suppression after alleviation of negative feedback. The unique mechanism of action of taselisib is most notable when comparing signaling suppression at 24 hours vs. 1 hour of drug exposure. In mutant cells, Taselisib displays greater pathway suppression at 24 hours and is more effective at maintaining pathway suppression upon re-activation of growth factor RTK signaling. This mechanism is specific to PIK3CA mutant cells and not observed in wildtype cells. Ongoing studies to further elucidate this mechanism of action will be presented. Citation Format: Kyle A. Edgar, Kyung Song, Stephen Schmidt, Don Kirkpatrick, Lilian Phu, Michelle Nannini, Rebecca Hong, Eric Cheng, Amy Young, Deepak Sampath, Lori S. Friedman. The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action. [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 370.

Abstract 242: Deciphering the role of PTEN as a predictive biomarker to next generation isoform-selective PI3K inhibitors in PIK3CA mutant breast cancer cells

Abstract The phosphoinositide-3 kinase (PI3K) pathway is one of the most frequently activated signaling pathways in human cancer and represents a promising therapeutic target. Taselisib (GDC-0032), a potent and selective inhibitor of the PI3K alpha, delta and gamma isoforms, but less potent in the beta isoform, displays increased sensitivity in models that are driven by mutant PIK3CA (Ndubaku CO et al, J Med Chem, 2013). Selective pressure caused by, small molecules such as PI3K inhibitors will ultimately lead to clinical resistance and disease progression. Recently, a published mechanism of acquired resistance to the PI3K inhibitor alpelisib (BYL-719, a PI3K alpha-selective inhibitor) reported that genomic loss of the tumor suppressor PTEN led to clinical progression in a metastatic breast cancer patient harboring an activating PIK3CA mutation (Juric D et al, Nature, 2014). As taselisib and alpelisib are next generation PI3K inhibitors, we sought to determine if there are any differences to drug sensitivity in PIK3CA mutant cells that have concomitant loss of PTEN. To investigate this hypothesis, we first measured viability in breast cancer cells, each with differing genetic backgrounds treated with two PI3K inhibitors, taselisib and alpelisib. We found that PIK3CA mutant and PIK3CA mutant/PTEN null cells were sensitive to treatment when compared to wild-type and PTEN null cells. However, PIK3CA mutant/PTEN null cells displayed higher IC50 values for both inhibitors when compared to PIK3CA mutant cells. To control for differences in genetic backgrounds, we next tested whether knockdown (KD) of PTEN expression in PIK3CA mutant breast cancer cells would promote resistance and reactivate the PI3K signaling cascade even in the presence of a PI3K inhibitor. Transient, siRNA-targeted KD of PTEN in the PIK3CA mutant breast cancer cell lines EFM19 and T47D conferred resistance to alpelisib after 6 days of drug treatment, an observation that was less pronounced in taselisib treated cells. Additionally, PTEN KD in DMSO-treated cells led to an increase in cell proliferation and enhanced downstream PI3K signaling as measured by phosphorylated AKT and S6 proteins when compared to non-targeting siRNA controls. The reactivated, pro-survival signaling with PTEN KD was maintained upon treatment with both inhibitors when compared to control cells, an observation that was more pronounced in alpelisib treated cells. These data suggest that PIK3CB can compensate for PIK3CA inhibition and that PTEN loss could lead to clinical resistance to alpelisib, but may not constitute a resistance mechanism to taselisib. As both molecules have entered phase 3 studies in estrogen receptor positive breast cancer, understanding how biomarker-defined genotypes respond to next generation PI3K inhibitors is critical for identifying patients most likely to gain therapeutic benefit. Citation Format: Heather M. Moore, Heidi M. Savage, Mark R. Lackner, Timothy R. Wilson. Deciphering the role of PTEN as a predictive biomarker to next generation isoform-selective PI3K inhibitors in PIK3CA mutant breast cancer cells. [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 242.

Abstract 2672: Characterization of the enhanced potency of PI3K inhibitor taselisib (GDC-0032) in PI3K mutant cell lines and models

Abstract Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene or loss of the tumor suppressor PTEN. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis; thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The current study investigates preclinical mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of PI3K alpha sparing inhibition of PI3K beta. In preclinical studies, taselisib demonstrates greater potency in cancer cell lines harboring activating PI3K alpha mutations vs wild-type lines. Taselisib induces tumor growth arrest and regressions at tolerated doses in xenograft and patient-derived xenograft (PDX) tumor models bearing PI3K alpha mutations. Notably, taselisib is distinguished from other PI3K inhibitors by enhanced potency in PI3K alpha mutant isogenic cells compared to parental cells. Pathway inhibition and apoptosis are associated with the enhanced activity of taselisib in PI3K alpha mutant cells. Other PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not achieve the same level of activity in PI3K alpha mutant cell lines. Taselisib is more effective at maintaining suppression of the signaling pathway upon activation of Receptor Tyrosine Kinases (RTKs), such as after alleviation of negative feedback within the pathway. Additionally, a screen to evaluate secreted factors contributing to pan-PI3K inhibitor resistance was carried out in SW48 parental and PI3Kα H1047R isogenic cells. Growth factor ligands for the EGFR family were found to reduce the activity of PI3K inhibitors. Taken together, taselisib offers greater preclinical activity in PI3K alpha mutant cells when compared to other PI3K inhibitors, which may indicate a wider therapeutic index. Citation Format: Kyle A. Edgar, Michelle Nannini, Rebecca Hong, Charlie Eigenbrot, Stephen Schmidt, Amy Young, Deepak Sampath, Jeffrey J. Wallin, Lori S. Friedman. Characterization of the enhanced potency of PI3K inhibitor taselisib (GDC-0032) in PI3K mutant cell lines and models. [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 2672. doi:10.1158/1538-7445.AM2015-2672