pan-PI3K Inhibitor Research Articles

Targeted Inhibition of the PI3K/Akt/mTOR Signaling Axis: Potential for Sarcoma Therapy.

Sarcoma is a heterogeneous group of malignancies often resistant to conventional chemotherapy and radiation therapy. The phosphatidylinositol-3-kinase/ protein kinase B /mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway has emerged as a critical cancer target due to its central role in regulating key cellular processes such as cell growth, proliferation, survival, and metabolism. Dysregulation of this pathway has been implicated in the development and progression of bone sarcomas (BS) and soft tissue sarcomas (STS). PI3K/Akt/mTOR inhibitors have shown promising preclinical and clinical activity in various cancers. These agents can inhibit the activation of PI3K, Akt, and mTOR, thereby reducing the downstream signaling events that promote tumor growth and survival. In addition, PI3K/Akt/mTOR inhibitors have been shown to enhance the efficacy of other anticancer therapies, such as chemotherapy and radiation therapy. The different types of PI3K/Akt/mTOR inhibitors vary in their specificity, potency, and side effect profiles and may be effective depending on the specific sarcoma type and stage. The molecular targeting of PI3K/Akt/mToR pathway using drugs, phytochemicals, nanomaterials (NMs), and microbe-derived molecules as Pan-PI3K inhibitors, selective PI3K inhibitors, and dual PI3K/mTOR inhibitors have been delineated. While there are still challenges to be addressed, the preclinical and clinical evidence suggests that these inhibitors may significantly improve patient outcomes. Further research is needed to understand the potential of these inhibitors as sarcoma therapeutics and to continue developing more selective and effective agents to meet the clinical needs of sarcoma patients.

Antiplatelet drug ticagrelor suppresses triple negative breast cancer metastasis by targeting PI3K

Metastatic recurrence is still a major challenge in breast cancer treatment. Patients with triple negative breast cancer (TNBC) develop early recurrence and relapse more frequently. Due to the lack of specific therapeutic targets, new targeted therapies for TNBC are urgently needed. Phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway is one of the active pathways involved in chemoresistance and survival of TNBC, being considered as a potential target for TNBC treatment. Our present study identified ticagrelor, an anti-platelet drug, as a pan-PI3K inhibitor with potent inhibitory activity against four isoforms of class I PI3K. At doses normally used in clinic, ticagrelor showed weak cytotoxicity against a panel of breast cancer cells, but significantly inhibited the migration, invasion and the actin cytoskeleton organization of human TNBC MDA-MB-231 and SUM-159PT cells. Mechanistically, ticagrelor effectively inhibited PI3K downstream mTOR complex 1 (mTORC1) and mTORC2 signaling by targeting PI3K and decreased the protein expression of epithelial-mesenchymal transition (EMT) markers. In vivo, ticagrelor significantly suppressed tumor cells lung metastasis in 4T1 tumor bearing BALB/c mice model and experimental lung metastasis model which was established by tail vein injection of GFP-labeled MDA-MB-231 cells. The above data demonstrated that ticagrelor can inhibit the migration and invasion of TNBC both in vitro and in vivo by targeting PI3K, suggesting that ticagrelor, a pan-PI3K inhibitor, might represent a promising therapeutic agent for the treatment of metastatic TNBC.

MDB-89. DETERMINING THE IMMUNOMODULATORY POTENTIAL OF PI3KΓ IN AGGRESSIVE MEDULLOBLASTOMA AND THE TUMOR MICROENVIRONMENT

Abstract BACKGROUND Medulloblastoma (MB) is the most common pediatric embryonal brain tumor arising in the cerebellum. It has a “cold” tumor microenvironment (TME) characterized by an abundance of immunosuppressive Tumor Associated Macrophages (TAMs) and low T-cell infiltration. TAMs include tissue resident microglia and migrating bone marrow derived macrophages. While microglia play important roles in the central nervous system through their immune surveillance and ability to activate other immune cells, they can be polarized by tumor cells toward an anti-inflammatory state which allows for maintained tumor growth and proliferation. A potential new target to combat the immunosuppressed TME of MB is the class IB isoform PI3Kγ. Inhibiting this isoform has been attributed to controlling a critical switch between immune suppression and immune stimulation in TAMs but has yet to be examined in MB and its TME. We hypothesize that inhibiting PI3Kγ in TAMs and MB tumor cells will induce an anti-tumorigenic immune response within the MB TME and promote tumor clearance. METHODS Engineered murine MB cells, mCB DNp53 MYC, are treated with a pan-PI3K or specific PI3Kγ inhibitor for 48 hours before being co-cultured with naïve splenic CD4 T-cells from C57BL/6J mice for 5 days. Proliferation of the T cells is measured using Cell Trace Violet through flow cytometry on a Cytek Aurora. RESULTS Pan-PI3K inhibition revealed that tumor cells treated with the inhibitor prior to co-culturing did not suppress T-cell proliferation as drastically when compared to untreated tumor cells. Treatment with a selective inhibitor is on-going. CONCLUSIONS These results suggest that inhibition of the PI3K pathway in our MB tumor cells dampens their ability to suppress T cell proliferation. The PI3K pathway may play an active role in tumor cell’s ability to induce immunosuppression. Inhibiting this pathway may be a new avenue for therapeutic intervention for relapsed/refractory MB.

Evaluation of Copanlisib in Combination with Eribulin in Triple-negative Breast Cancer Patient-derived Xenograft Models.

In this research, we demonstrated that the pan-PI3K inhibitor copanlisib enhanced the cytotoxicity of eribulin in a panel of TNBC PDX models. The improved tumor growth inhibition was irrespective of PI3K pathway alteration and was corroborated by the enhanced mitotic arrest and apoptotic induction observed in PDX tumors after combination therapy compared with each drug alone. These data provide the preclinical rationale for the clinical testing in TNBC.

Combined inhibition of class 1-PI3K-alpha and delta isoforms causes senolysis by inducing p21WAF1/CIP1 proteasomal degradation in senescent cells

The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21WAF1/CIP1 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies.

Abstract GS03-10: Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations

Abstract Background: PIK3CA mutations occur in ~40% of HR-positive breast cancers, where alpelisib, an orthosteric PI3Kα inhibitor, is FDA-approved in combination with fulvestrant. Although prior studies have identified potential resistance mechanisms, such as PTEN loss, clinical acquired resistance to orthosteric PI3Kα inhibitors and the role of next-generation allosteric PI3Kα inhibitors remain poorly understood. Methods: To identify on-target and off-target alterations potentially mediating resistance to PI3Kα inhibitors, we used a targeted next-generation sequencing assay (Guardant360; Guardant Health) to analyze ctDNA in serially collected plasma samples from 32 patients with PIK3CA-mutated advanced HR-positive, HER2-negative breast cancer treated with alpelisib and inavolisib. In addition, we performed whole exome sequencing (WES) of 100 tissue samples collected from 8 autopsy series from patients with metastatic, PIK3CA-mutant HR-positive, HER2-negative breast cancer previously treated with PI3Kα inhibitors. Acquired alterations were prioritized through a combination of structural modeling and free-energy perturbation simulation and validated in genomically engineered PIK3CA mutant breast cancer cell lines T47D (PIK3CA H1047R-mutant) or MCF7 (PIK3CA E545K-mutant). Results: We observed that 50% of patients acquire genomic alterations within the PI3K-pathway, including PTEN loss and activating AKT1 mutations. Notably, while secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Ka-inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket including PIK3CA Q859K and PIK3CA W780R. Some mutations had differential effects on PI3Ka-selective vs. pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Ka-inhibitor RLY-2608. Conclusion: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Ka inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Ka inhibitor. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers. Citation Format: Andreas Varkaris, Ferran Fece de la Cruz, Elizabeth Martin, Bryanna Nordern, Nicholas Chevalier, Ignaty Leshchiner, Anastasia Stavridi, Janice Kim, Alkistis Papatheodoridi, Hakan Gunaydin, Brian Danysh, Laxmi Parida, Ioannis Sanidas, Yongli Ji, Kayao Lau, Gerburg Wulf, Aditya Bardia, Laura Spring, Steven Isakoff, Jochen Lennerz, Levi Pierce, Ermira Pazolli, Gad Getz, Ryan Corcoran, Dejan Juric. Allosteric PI3K-alpha inhibition overcomes on-target resistance to orthosteric inhibitors mediated by secondary PIK3CA mutations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-10.

Abstract LB449: Strategies to overcome resistance to PI3Kalpha-selective inhibitors mediated by acquired alterations in the PI3K/AKT pathway

Abstract PIK3CA is among the most frequently mutated kinases in multiple types of cancer, including in 40% of hormone receptor-positive, HER2-negative breast cancers. The PI3Kα-selective orthosteric inhibitor alpelisib is the only drug targeting PIK3CA currently approved in this patient population, in combination with fulvestrant. As commonly observed with other targeted therapies, resistance often develops during treatment with PI3Kα-selective inhibitors. In one of the largest patient cohorts to date, including serial liquid biopsies and rapid autopsies in 39 patients, our group has observed genomic alterations within the PI3K/AKT pathway as one of the major mechanisms of resistance to these agents. Among them, we have identified for the first-time secondary mutations in PIK3CA (W780R and Q859K) that decrease the affinity of the PI3Kα-selective inhibitors alpelisib and inavolisib, leading to acquired resistance. Multiple small molecule inhibitors designed to suppress signaling through the PI3K/AKT axis are currently in pre-clinical development. These include AKT inhibitors, isoform-selective as well as pan-PI3K inhibitors, and a novel subclass of allosteric PI3Kα-selective inhibitors designed to preferentially inhibit both kinase (H1047R) and helical (E545K) domain mutant PI3Kα activity. We have introduced all the emergent alterations observed in our patient cohort into breast cancer models and screen how they modify the effect of a structurally diverse array of PI3K/AKT inhibitors. While some acquired mutations compromise the effect of specific drugs, we have determined that the W780R mutation could drive universal resistance to ATP-competitive PI3K inhibitors that bind the catalytic pocket. Importantly, vertical pathway inhibition or allosteric inhibition with pan-mutant-selective PI3Kα inhibitors such as RLY-2608 or STX-478 could overcome resistance induced by all emerging secondary PIK3CA mutations.Dose-limiting toxicity in the form of hyperglycemia, rash or gastrointestinal issues are common in patients treated with orthosteric PI3Kα inhibitors. While mutant selective PI3Kα inhibitors induce less adverse effects in patients, they have a decreased potency targeting wildtype PIK3CA. We have identified wildtype PIK3CA-mediated feedback pathway reactivation as a potential mechanism of resistance to this type of inhibitors. Our work suggests that combining orthosteric and allosteric PI3Kα inhibitors as well as downstream pathway deactivation with AKT inhibitors could result in an increased therapeutic index and delay the emergence of resistance in patients. This work provides an insightful characterization of the current knowledge on clinical acquired resistance to PI3Kα inhibitors and proposes a detailed strategy to overcome resistance mediated by most PI3K/AKT alterations described to date. Citation Format: Ferran Fece de la Cruz, Andreas Varkaris, Elizabeth E. Martin, Bryanna L. Norden, Nicholas Chevalier, Allison M. Kehlmann, Ignaty Leshchiner, Haley Barnes, Sara Ehnstrom, Parasvi Patel, Janice S. Kim, Haley Ellis, Ioannis Sanidas, Kayao T. Lau, Aditya Bardia, Laura M. Spring, Steven J. Isakoff, Jochen K. Lennerz, Gad Getz, Ryan B. Corcoran, Dejan Juric. Strategies to overcome resistance to PI3Kalpha-selective inhibitors mediated by acquired alterations in the PI3K/AKT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB449.

Abstract CT042: NCI10217: Phase Ib biomarker-driven tumor-agnostic combination (Combo) trial of copanlisib (Copa) and olaparib (Ola) in molecularly-selected patients (pts) with advanced cancers with PIK3CA hotspot, PTEN and DNA damage response (DDR) mutations

Abstract Background: Preclinically, PI3K and PARP inhibition synergize in cancers with DDR and PI3K pathway alterations. NCI10217 assessed the combo of Copa (pan-PI3K inhibitor) and Ola (PARP inhibitor) in tumors harboring mutations in PI3K/AKT or DDR pathways. Materials and Methods: Eligible pts had tumors with pathogenic PIK3CA hotspot, PTEN and DDR mutations. Copa was administered at 45 mg to 60 mg IV on Days (D) 1 and 15 or 1, 8 and 15 with Ola at 200 mg to 300 mg twice daily (BID) orally in 28-days cycles. Prior PARP and PI3K inhibitors were permitted. Fresh tumor biopsies and ctDNA were mandated at baseline and C1D15 and optional at the end of trial. Study objectives were safety, RP2D, pharmacokinetics, antitumor activity and translational analyses of serial tumor (WES; RNA-Seq; RPPA) and ctDNA samples. Results: 28 pts (M:F 8:20; ECOG PS 0:1 9:19; mean age 58y (28-76y) with breast (n=6), colorectal (n=5), prostate (n=4), endometrial (n=2), gastric (n=2), vaginal (n=2), appendix, cervix, head and neck, leiomyosarcoma, ovarian, peritoneal, and renal (n=1) cancers were enrolled. Pts with BRCA1/2 (n=12), PTEN (n=10), PIK3CA hotspot (n=6), ARID1A (n=4), CHEK2 (n=3), PALB2 (n=3), ATM (n=2), CDK12, CHEK1, MRE11A, RAD51C (n=1 each) mutations were enrolled. 10 pt tumors harbored >1 qualifying mutation. 17 (61%)/28 pts had ≥3 prior lines of therapy; 2 (7%)/28 had prior PARP inhibitors and 1 (4%)/28 had prior PI3K inhibitor. Common treatment-related toxicities included G1/2 mucositis (43%), nausea (43%), diarrhea (36%), anorexia (29%), fatigue (29%), maculo-papular rash (29%), hyperglycemia (25%), vomiting (25%); G3 transient hypertension (29%). Dose-limiting toxicities occurred in 3 (11%)/28 pts: 1 pt (G3 elevated transaminases) at Copa 45 mg (D1, D8 and D15) + Ola 200 mg BID, and 2 pts (G2 mucositis and maculo-papular rash, and G4 hyperglycemia) at Copa 60 mg (D1, D8, and D15) + Ola 300 mg BID. Of 22 pts evaluable for RECISTv1.1 criteria, 6 (27%)/22 pts achieved RECISTv1.1 partial responses (PR): 2 pts with BRCA1 mutation breast cancer; 1 pt with PIK3CA + PTEN + ARID1A mutation breast cancer; 1 pt with PTEN + PALB2 mutation breast cancer and prior PARP inhibitor exposure; 1 pt with BRCA2 mutation prostate cancer; 1 pt with BRCA2 + PTEN mutation prostate cancer. 8 (36%)/22 pts achieved RECISTv1.1 stable disease (SD); 2 (25%)/8 pts had SD≥6 months, including a pt with CA125 response (>50% reduction). Clinical benefit rate (SD≥6 months + PR) was 36%. Responses were deep (up to -100%) and durable (up to 37+ months). Based on these data, the RP2D was established at Copa 60 mg D1 and D15 + Ola 300 mg BID. Conclusion: Copa + Ola was safe and well-tolerated with clinical benefit observed in 36% of pts with advanced cancers with PIK3CA hotspot, PTEN and/or DDR mutations. Translational analyses are ongoing. Citation Format: Timothy A. Yap, Ecaterina E. Dumbrava, Jordi Rodon Ahnert, Apostolia M. Tsimberidou, David Hong, Sarina A. Piha-Paul, Daniel D. Karp, Siqing Fu, Aung Naing, Paula Pohlmann, Jessica R. Rhudy, Sheila B. Berenji-Jalaei, Ashley A. Cole, Christian Valladolid, Desirae Dufner, Bruno B. Bockorny, Andrea Bullock, Daniel Fein, Elizabeth Buchbinder, Atish Choudhury, Candace Haddox, Elizabeth Lee, Gerburg Wulf, Percy Ivy, Rabih Said, Geoffrey Shapiro, Funda Meric-Bernstam. NCI10217: Phase Ib biomarker-driven tumor-agnostic combination (Combo) trial of copanlisib (Copa) and olaparib (Ola) in molecularly-selected patients (pts) with advanced cancers with PIK3CA hotspot, PTEN and DNA damage response (DDR) mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT042.

Abstract 1230: MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance

Abstract Adaptive resistance mechanisms compromise the long-term effectiveness of kinase-targeted agents dictating the need for strategic combination approaches. MTX-531 was computationally designed to selectively target both PI3K and wild type EGFR. MTX-531 exhibits low nanomolar potency against the PI3K isoform family and EGFR with a high degree of specificity predicted by co-crystal structural analyses. Full kinome screening confirmed that MTX-531 is exquisitely selective for its intended targets. We evaluated the pharmacological profile of MTX-531, whereupon single agent treatment with this lead molecule elicited a high incidence of durable tumor regressions in a broad panel of PIK3CA mutant CDX and PDX squamous head and neck (HNSCC) models. The combination of MTX-531 with RAS pathway inhibitors, including agents directed against KRAS G12C, led to durable regressions of KRAS mutant colorectal and pancreatic xenografts, resulting in striking increases in median survival. MTX-531 is exceptionally well tolerated in mice and uniquely does not lead to hyperglycemia and hyperinsulinemia commonly seen with predecessor pan PI3K inhibitors. MTX-531 acts as an agonist of PPARγ, a structural feature of the molecule that is thought to mitigate hyperglycemia induced by PI3K inhibition. This unique attribute of MTX-531 confers a favorable therapeutic index not typically seen with PI3K inhibitors. To the best of our knowledge, MTX-531 is the first reported pan-PI3K inhibitor that does not lead to hyperglycemia. The pharmaceutical profile of MTX-531 includes favorable cross-species oral bioavailability, microsomal stability, and a facile 3 step chemical synthesis. MTX-531, which has the unique capability of concurrently and selectively inhibiting PI3K and EGFR, illustrates the power of rational computational drug design to target multiple adaptive resistance mechanisms in a single molecule. The versatility of MTX-531 in both the single agent and combination settings offers a breadth of development strategies that continue to evolve as new combination candidates become available. MTX-531 is currently undergoing advanced preclinical development for anticipated first in human clinical trials in late 2024. Citation Format: Christopher E. Whitehead, Elizabeth K. Ziemke, Christy L. Frankowski-McGregor, Rachel A. Mumby, June Chung, Jinju Li, Nathaniel Osher, Oluwadara Coker, Michelle Norris, Scott Kopetz, Veera Baladandayuthapani, Melinda Hollingshead, Sharad Verma, Judith S. Sebolt-Leopold. MTX-531, a first-in-class pan-PI3K inhibitor spares hyperinsulinemia yielding durable tumor regressions and resilience to adaptive resistance [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 1230.

Abstract 4608: Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids

Abstract The alternative splicing of mRNA precursors allows one gene the capacity to yield multiple protein isoforms, each with distinct functions. Although this intricate process is subject to stringent regulation under normal physiological conditions, aberrant alternative splicing can generate atypical proteins and contribute to various diseases, including cancer. By activating splicing factors, Cdc-like kinases (CLKs) serve as pivotal regulators of alternative splicing and are considered promising therapeutic targets for various tumors. In this study, we investigated the activity of two CLK inhibitors, cirtuvivint (SM08502) and CC-671, in combination with precision oncology agents or conventional chemotherapeutics. Thirty highly characterized patient-derived cancer cell lines were selected from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm). The cell lines were derived from a range of cancer types including head and neck, bladder, pancreas, endometrial, colon, and melanoma, and contained clinically relevant variants of KRAS (G12C, G12D). Multi-cell type tumor spheroids were grown from a ratio of 6:2.5:1.5 malignant cells, endothelial cells, and mesenchymal stem cells, respectively. Following three days of growth, the spheroids were treated with single agents or combinations at concentrations up to their clinical Cmax value, if reported. After seven days of continuous drug exposure, cell viability was assessed using the CellTiter-Glo 3D assay. As a single agent, cirtuvivint showed concentration-dependent activity in all spheroid types with 1 – 3 logs of cytotoxicity, whereas CC-671 did not show activity in all spheroid types and achieved no more than 1 log of cytotoxicity. Among the standard chemotherapeutic drugs, doxorubicin and SN-38 demonstrated additive and greater-than-additive cytotoxicity in various spheroid types when combined with either CLK inhibitor. Several of the targeted oncology drugs exhibited noteworthy additive and greater-than-additive cytotoxicity when combined with a CLK inhibitor. These agents included the XPO1 inhibitor, eltanexor, the MCL-1 inhibitor, tapotoclax, the α-isoform-specific PI3K inhibitor, inavolisib, and the pan-PI3K inhibitor, copanlisib. Notably, combinations of the CLK inhibitors with the KRAS G12D variant-specific inhibitor, MRTX-1133, showed selective activity against all tumor cell lines harboring this genetic variant. Interestingly, a strong antagonistic interaction between paclitaxel and CC-671 was observed in all thirty spheroid types, while no such interaction was observed with cirtuvivint. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Nathan P. Coussens, Steven D. Gore, James H. Doroshow, Beverly A. Teicher. Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids [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 4608.

Abstract 1452: A PIK3CA-driven mouse model of metaplastic breast carcinoma to assess potential therapeutic strategies

Abstract Metaplastic Breast Carcinomas (MBC) are rare and aggressive breast cancers with mesenchymal or squamous components. While histologically heterogeneous, MBC are typically triple negative: they lack expression of ER, PR and HER2. Importantly, approximately 40% of MBC harbor oncogenic gain-of-function mutations in PIK3CA, the gene encoding for the catalytic subunit of PI3K. These alterations are targetable by an array of inhibitors of the PI3K-AKT-mTORC1 pathway. However, few animal models of MBC are available, and it is currently difficult to study MBC biology and response to therapy. Here, we describe the generation of a new mouse model of MBC driven by the PIK3CA H1047R mutation. We histologically and biologically characterize this model and assess potential therapeutic strategies. We used genetically-engineered mouse models to express the human PIK3CA H1047R mutation under the control of the MMTV promoter, which is mostly expressed in the mammary epithelium. Tumors developed in mice in about 1 year. We histologically characterized these tumors and generated cell lines, organoids, and tumor grafts in immunocompromised mice. The resulting model, termed MMTV-102, was evaluated for the response to PI3K-AKT-mTOR pathway inhibitors, both in vitro and in vivo. The MMTV-PIK3CAH1047R tumor was a high grade poorly differentiated MBC negative for ER, PR, and HER2. It presented squamous components, keratin depositions, mitotic figures, and vascular invasion and stained positive for the basal marker CK14. In addition, due to the presence of the PIK3CA H1047R mutation, it stained positive for phosphorylated AKT, PRAS40, and S6RP, indicating constitutive activation of the PI3K-AKT-mTORC1 pathway. MBC derived cell lines and organoids were confirmed to be triple negative and could form tumors in mice. In addition, these cellular models were sensitive to PI3Kα, pan-PI3K, and mTORC1 inhibitors in vitro and in vivo, but the mTORC1 inhibitor everolimus exhibited the strongest effect. When resistance to everolimus eventually developed in vivo, we established cell lines from these resistant tumors. In addition, we generated an everolimus resistant cell line following chronic drug exposure in vitro (MMTV-102 Par res). Both cellular models were tested for their sensitivity to the next-generation mTORC1 inhibitor RMC-5552 by proliferation and clonogenic assays. We show that our mouse model can originate MBC with an active PI3K pathway. These tumors are amenable to transplantation into immunocompromised mice and to generate cell lines and organoids, allowing us to develop a simple, versatile, and rapid model of MBC. Tumors derived from our model are sensitive to PI3Kα and mTORC1 inhibitors, but eventually develop resistance to these agents. The use of next-generation mTORC1 inhibitors can overcome resistance in some cases, providing a rationale for their implementation in the treatment of patients with MBC. Citation Format: Paola Roa, Valentina Foglizzo, Michelle R. de Marchena, Shraddha Chandthakuri, Emiliano Cocco, Pau Castel. A PIK3CA-driven mouse model of metaplastic breast carcinoma to assess potential therapeutic strategies [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 1452.

Abstract 582: Oncogenic PIK3CA fortifies immune determinants in vascular cancers

Abstract Angiosarcomas are a group of vascular cancers that form malignant blood vessels. These malignancies are seemingly inflamed primarily due to their pathognomonic nature, which consists of irregular endothelium and tortuous blood channels. The malignant vascular spaces host a variety of blood and immune components, where a controlled scavenging system is necessary for tumor maintenance. Oncogenes may convey molecular signals to foster the tumor immune niche, and PIK3CA mutations that induce continuous activation of the PI3K pathway occur in a subset of angiosarcomas. Despite the undoubtedly oncogenic properties of PIK3CA mutations, the mechanistic role of these mutations - particularly the potential for controlling the hemato-endothelial system in angiosarcomas - remains undetermined. In this study, we demonstrate the functional consequences of oncogenic PIK3CA and its molecular properties in angiosarcoma cells. We created isogenic cell clones from two angiosarcoma cell lines engineered to harbor hotspot mutations in PIK3CA using CRISPR/Cas9: two homozygous PIK3CA H1047R cell clones (C8 and C35) and one wild-type (WT) clone were generated from DHSA-1426 angiosarcoma cells; and one homozygous mutant clone was created from COSB cells. We found that PIK3CA mutant cells constitutively activated the PI3K/AKT/mTOR pathway at a greater level than that of WT cells. Bulk- and single cell(sc)-RNA-seq data revealed that mutant cells enriched gene sets associated with PI3K signaling and other oncogenic pathways including KRAS, MAPK, and TGF-beta. Also, mutant cells significantly increased cytokines regulating immune cell migration and chemotaxis such as IL-8 and MCP-1. In addition to the common features, we identified functional and molecular divergence between mutant clones. Noticeable changes in cellular morphology, growth kinetics, and tube-forming capacity were observed between two mutant clones. C35 mutant cells were more sensitive to both pan-PI3K and PI3K-alpha specific inhibitor, and DNA damage was greater than in the C8 clone. C35 cells enhanced activation of IL-6/STAT3 signaling compared to C8, despite the increment of the aforementioned immune cytokines in both mutants. Moreover, our comprehensive analysis of bulk- and sc-RNA-seq, ATAC-seq, and metabolomics data identified molecular signatures associated with metabolic vulnerabilities of C35 cells. In summary, we demonstrate that oncogenic PIK3CA perpetuates PI3K signaling activation and reinforces a supply for immune signaling. Furthermore, our data suggest that PIK3CA mutations contribute to establishing cellular heterogeneity that reveals a spectrum of vascular, immunogenic, and metabolic activities. Our ongoing work involves developing an experimental model of angiosarcoma using induced pluripotent stem cells to examine if oncogenic PIK3CA controls immune determinants during malignant endothelial transformation. Citation Format: Donghee Lee, Emma Kozurek, Md Abdullah, Rong Li, Erin B. Dickerson, Jong Hyuk Kim. Oncogenic PIK3CA fortifies immune determinants in vascular cancers [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 582.

Abstract 1924: RTK signaling and WT RAS activity as vulnerabilities in tumors with acquired resistance to GDP-state selective KRASG12C inhibitors in preclinical models

Abstract Background: KRASG12C-GDP Inhibitors such as sotorasib and adagrasib have demonstrated clinical benefit in lung cancer patients harboring an oncogenic KRASG12C mutation. However, the durability of monotherapy benefit is limited by the development of resistance. Hence, identification of novel treatment approaches to overcome resistance and extend duration of benefit becomes necessary to address this unmet clinical need. Experimental Design: We generated two NSCLC models of KRASG12C−GDP inhibitor acquired resistance, named sotorasib-R LU65 (cell-line derived, in vitro) and adagrasib-R LUN156 (PDX-derived, in vivo) following extended treatment with sotorasib and adagrasib, respectively. We then performed transcriptomic and mass spectrometry-based phosphoproteomics analyses on baseline and KRASG12C-GDP inhibitor-resistant samples to determine resistance and escape mechanisms. Results: In the sotorasib-R LU65 cells, we discovered upregulation of various RTKs including HER2, HER3, and AXL using a mass spectrometry-based proteomics approach and Immunoblot analysis. Simultaneously, from a transcriptome analysis, we observed activation of RAS and PI3K/AKT signaling in these cells. In the adagrasib-R LUN156 model, increased phosphorylation of MET was observed via phospho-RTK array and by immunoblotting. Transcriptomic analysis revealed an enrichment for RAS pathway dependency in the adagrasib-R tumor models. RAS GTP-binding assessed by RAF-RBD pulldown and multiple reactions monitoring mass-spectrometry suggest increased GTP-bound RAS isoforms in sotorasib-R LU65 cells and active RAS signaling in adagrasib-R LUN156. Given the convergence of our findings on re-activation/maintenance of wild-type RAS signaling as a means of resistance to KRASG12C-GDP-state inhibitors, we hypothesized that RMC-7977, a tri-complex RASMULTI-GTP inhibitor, which inhibits signaling via mutant and wild-type RAS, had the potential to counteract these mechanisms. We tested RMC-7977 monotherapy, which drove complete tumor regressions in the adagrasib-R LUN156 model. Additionally, RMC-7977 attenuated tumor growth in sotorasib-R cell derived LU65 xenograft model wherein we observed co-activation of PI3K/AKT signaling. Thus, we tested a combination with the pan-PI3K inhibitor pictilisib and observed combinatorial activity consistent with our findings. Conclusions: These data suggest that in KRASG12C-GDP inhibitor-resistant models RTK activation maintains MAPK dependency and in the case of one model, PI3K signaling. Consistent with these mechanisms, RMC-7977 as a single agent or in combination with pictilisib drove significant tumor regressions in these models. These preclinical results indicate RASMULTI-GTP inhibition alone or combination with PI3K inhibition has the potential to address KRASG12C-GDP inhibitor resistance. Citation Format: Hitendra S. Solanki, Harshit Shah, Denis Imbody, Bina Desai, Paul A. Stewart, Bin Fang, Yaakov Stern, Lancia N. Darville, John M. Koomen, Andriy Marusyk, Ethan Ahler, Ida Aronchik, Mallika Singh, Eric B. Haura. RTK signaling and WT RAS activity as vulnerabilities in tumors with acquired resistance to GDP-state selective KRASG12C inhibitors in preclinical models [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 1924.

A novel pan-PI3K inhibitor KTC1101 synergizes with anti-PD-1 therapy by targeting tumor suppression and immune activation

BackgroundPhosphoinositide 3-kinases (PI3Ks) are critical regulators of diverse cellular functions and have emerged as promising targets in cancer therapy. Despite significant progress, existing PI3K inhibitors encounter various challenges such as suboptimal bioavailability, potential off-target effects, restricted therapeutic indices, and cancer-acquired resistance. Hence, novel inhibitors that overcome some of these challenges are needed. Here, we describe the characterization of KTC1101, a novel pan-PI3K inhibitor that simultaneously targets tumor cell proliferation and the tumor microenvironment. Our studies demonstrate that KTC1101 significantly increases the anti-PD-1 efficacy in multiple pre-clinical mouse models.MethodsKTC1101 was synthesized and characterized employing chemical synthesis, molecular modeling, Nuclear Magnetic Resonance (NMR), and mass spectrometry. Its target specificity was confirmed through the kinase assay, JFCR39 COMPARE analysis, and RNA-Seq analysis. Metabolic stability was verified via liver microsome and plasma assays, pharmacokinetics determined by LC–MS/MS, and safety profile established through acute toxicity assays to determine the LD50. The antiproliferative effects of KTC1101 were evaluated in a panel of cancer cell lines and further validated in diverse BALB/c nude mouse xenograft, NSG mouse xenograft and syngeneic mouse models. The KTC1101 treatment effect on the immune response was assessed through comprehensive RNA-Seq, flow cytometry, and immunohistochemistry, with molecular pathways investigated via Western blot, ELISA, and qRT-PCR.ResultsKTC1101 demonstrated strong inhibition of cancer cell growth in vitro and significantly impeded tumor progression in vivo. It effectively modulated the Tumor Microenvironment (TME), characterized by increased infiltration of CD8+ T cells and innate immune cells. An intermittent dosing regimen of KTC1101 enhanced these effects. Notably, KTC1101 synergized with anti-PD-1 therapy, significantly boosting antitumor immunity and extending survival in preclinical models.ConclusionKTC1101's dual mechanism of action—directly inhibiting tumor cell growth and dynamically enhancing the immune response— represents a significant advancement in cancer treatment strategies. These findings support incorporating KTC1101 into future oncologic regimens to improve the efficacy of immunotherapy combinations.

Metformin and buparlisib synergistically induce apoptosis of non-small lung cancer (NSCLC) cells via Akt/FoxO3a/Puma axis

Non-small cell lung cancer (NSCLC) is a global health issue lacking effective treatments. Buparlisib is a pan-PI3K inhibitor that shows promising clinical results in treating NSCLC. However, chemoresistance is inevitable and hampers the application of buparlisib. Studies show that a combination of phytochemicals and chemotherapeutics enhances its effectiveness. Here, we evaluated the role of metformin, an agent with multiple pharmacological properties, in enhancing the anti-tumour activities of buparlisib against NSCLC cells. Our results showed that metformin and buparlisib synergistically inhibited cell viability, migration, and invasion of NSCLC cells. In addition, co-treatment of metformin and buparlisib also induced cell cycle arrest and cell death in NSCLC cells. Mechanistically, metformin and buparlisib repressed Mcl-1 and upregulated Puma in NSCLC cells in a p53-independent manner. Moreover, they inhibited the PI3K/Akt signalling pathway, leading to activation of the FoxO3a/Puma signalling in NSCLC cells. Our findings suggest that combined treatment of metformin and buparlisib might provide a promising strategy for treating NSCLC.

Differential modulation of PI3K/Akt/mTOR activity by EGFR inhibitors: A rationale for co-targeting EGFR and PI3K in cisplatin-resistant HNSCC.

To find a new strategy to treat cisplatin-resistant head and neck squamous cell carcinoma (HNSCC), we investigated the effects of EGFR inhibitors on the PI3K/Akt/mTOR pathway and determined the efficacy of EGFR inhibitors in combination with PI3K inhibitors to suppress cell proliferation in cisplatin-resistant-HNSCC. The cisplatin-resistant HNSCC cell lines were treated with four FDA approved EGFR inhibitors, which included Gefitinb or Erlotinib alone, or in combination with the pan-PI3K inhibitor, BKM120. Phosphorylation and total protein levels of cells were assessed by Western blot analysis. Cell proliferation was examined by MTS assay. Apoptosis was analyzed by flow cytometry. Cisplatin-resistant HNSCC cells were also resistant to EGFR inhibitors. However, a combination of EGFR inhibitors with PI3K inhibitor BKM120 dramatically improved the efficacy of EGFR inhibitors to inhibit cell proliferation and induce apoptosis. Furthermore, treatment with EGFR inhibitors differentially affected the phosphorylation of Akt and mTOR, which included partial inhibition, no inhibition, and induction. A combination of EGFR inhibitors and BKM120 completely blocked phosphorylation of EGFR, Akt, and S6K (an mTOR target). Our data provided a rationale for EGFR inhibitors in combination with PI3K inhibitors to treat cisplatin-resistant HNSCC.

Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

The gene expression profile and cell of origin of canine peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) refers to a heterogenous group of T-cell neoplasms with poor treatment responses and survival times. Canine PTCL clinically and immunophenotypically resembles the most common human subtype, PTCL-not otherwise specified (PTCL-NOS), leading to interest in this canine disease as a naturally occurring model for human PTCL. Gene expression profiling in human PTCL-NOS has helped characterize this ambiguous diagnosis into distinct subtypes, but similar gene expression profiling in canine PTCL is lacking.MethodsBulk RNA-sequencing was performed on tumor samples from 33 dogs with either CD4+ (26/33), CD8+ (4/33), or CD4-CD8- (3/33) PTCL as diagnosed by flow cytometry, and sorted CD4+ and CD8+ lymphocytes from healthy control dogs. Following normalization of RNA-seq data, we performed differential gene expression and unsupervised clustering methods. Gene set enrichment analysis was performed to determine the enrichment of canine CD4+ PTCL for human PTCL-NOS, oncogenic pathways, and various stages of T-cell development gene signatures. We utilized gene set variation analysis to evaluate individual canine CD4+ PTCLs for various human and murine T-cell and thymocyte gene signatures. Cultured canine PTCL cells were treated with a pan-PI3K inhibitor, and cell survival and proliferation were compared to DMSO-treated controls. Expression of GATA3 and phosphorylated AKT was validated by immunohistochemistry.ResultsWhile the canine CD4+ PTCL phenotype exhibited a consistent gene expression profile, the expression profiles of CD8+ and CD4-CD8- canine PTCLs were more heterogeneous. Canine CD4+ PTCL had increased expression of GATA3, upregulation of its target genes, enrichment for PI3K/AKT/mTOR signaling, and downregulation of PTEN, features consistent with the more aggressive GATA3-PTCL subtype of human PTCL-NOS. In vitro assays validated the reliance of canine CD4+ PTCL cells on PI3K/AKT/mTOR signaling for survival and proliferation. Canine CD4+ PTCL was enriched for thymic precursor gene signatures, exhibited increased expression of markers of immaturity (CD34, KIT, DNTT, and CCR9), and downregulated genes associated with the T-cell receptor, MHC class II associated genes (DLA-DQA1, DLA-DRA, HLA-DQB1, and HLA-DQB2), and CD25.ConclusionsCanine CD4+ PTCL most closely resembled the GATA3-PTCL subtype of PTCL-NOS and may originate from an earlier stage of T-cell development than the more conventionally posited mature T-helper cell origin.

A high affinity pan-PI3K binding module supports selective targeted protein degradation of PI3Kα.

Class I phosphoinositide 3-kinases (PI3Ks) control cellular growth, but are also essential in insulin signaling and glucose homeostasis. Pan-PI3K inhibitors thus generate substantial adverse effects, a reality that has plagued drug development against this target class. We present here evidence that a high affinity binding module with the capacity to target all class I PI3K isoforms can facilitate selective degradation of the most frequently mutated class I isoform, PI3Kα, when incorporated into a cereblon-targeted (CRBN) degrader. A systematic proteomics study guided the fine tuning of molecular features to optimize degrader selectivity and potency. Our work resulted in the creation of WJ112-14, a PI3Kα-specific nanomolar degrader that should serve as an important research tool for studying PI3K biology. Given the toxicities observed in the clinic with unselective PI3Kα inhibitors, the results here offer a new approach toward selectively targeting this frequently mutated oncogenic driver.

Class IA PI3K isoforms lead to differential signalling downstream of PKB/Akt

Abstract Objectives The catalytic subunits of Class IA PI3K, p110α, p110β, and p110δ, phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3) on the plasma membrane. In cancer, these catalytic subunits are usually found to be altered or amplified. Because pan-PI3K inhibition results in systemic toxicities, finding specific targets for the ubiquitous PI3K isoforms offers considerable potential for enhancing the effectiveness of PI3K-targeted therapy. Methods We aim to delineate the isoform-specific druggable targets of the PI3K by deleting PIK3CA (encoding p110α) and PIK3CB (encoding p110β) by Cre mediated excision and ectopically expressing p110α, p110β, or p110δ with or without myristoylation (Myr) tag in mouse embryonic fibroblasts (MEFs). Myr is a lipidation signal that translocates proteins to plasma membrane permanently. This translocation renders p110s constitutively activated as they remain in close proximity to PIP2 on the membrane. Results Unique and redundant Akt targets are identified downstream of different PI3K isoforms. mTORC1, one of the targets of fully-activated Akt, has been observed to be differentially regulated in MEFs upon expression of p110α or p110β. The varying dependencies on mTORC1 and Rac1 led us to analyse a potential scaffolding function of p110β with Rac1 to mediate phosphorylation and activation of mTOR using platforms for the modeling of biomolecular complexes. We also documented that p110α and p110β support cell cycle kinetics differentially. Conclusions This study suggests differential regulation of protein translation, metabolism, cell cycle, and survival signaling downstream of unique p110 targets, underlying the importance of cancer treatment according to the deregulated p110 isoform.