PIK3CA H1047R Research Articles

Pyrotinib and trastuzumab combination treatment synergistically overcomes HER2 dependency in HER2-positive breast cancer: insights from the PHILA trial

The PHILA study suggests that pyrotinib, trastuzumab, and docetaxel significantly improved progression-free survival (PFS) compared with placebo, trastuzumab, and docetaxel in patients with untreated HER2-positive metastatic breast cancer. In this study, we aimed to investigate the synergistic mechanisms of pyrotinib plus trastuzumab and provide further insights for the PHILA trial. The invitro activity of combination treatments was assessed through cell biological and biochemical experiments. The invivo efficacy was evaluated in cell-derived xenografts, a TUBO tumour model, and one clinical case. Next-generation sequencing was performed on circulating tumour DNA (ctDNA) from patients in the PHILA trial. The combination of pyrotinib and trastuzumab more effectively inhibited cell growth than pyrotinib or trastuzumab alone in models of HER2-dependent breast cancer. It potentiated membrane HER2 ubiquitination and downregulation, which resulted in a comprehensive blockade of the HER2 signalling pathway. The pyrotinib-altered membrane HER2 levels had no significant effect on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). We further validated the synergistic mechanisms in TUBO tumours and one clinical case, rather than models of HCC1954cells harbouring the PIK3CA H1047R mutation. Similarly, in our centre cohort of the PHILA study, patients with genetic alterations in the HER2 signalling cascade had significantly shorter median PFS than individuals with the wild-type pathway. Our findings underscore the robust synergy between pyrotinib and trastuzumab in overcoming HER2 dependency and provide a rationale for pyrotinib, trastuzumab, and docetaxel as one of the optimal choices for patients with untreated HER2-positive metastatic breast cancer, who are dependent on the HER2 signalling cascade. This work was supported by the National Key Research and Development Program of China (2021YFF1201300), the National Natural Science Foundation of China (82172875), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2022-I2M-2-001), and the Joint Innovative Fund of Beijing Natural Science Foundation and Changping District (L234004).

Identification of breast cancer-associated PIK3CA H1047R mutation in blood circulation using an asymmetric PCR assay.

To establish a highly sensitive and specific approach for the detection of circulating PIK3CA H1047R mutation in breast cancer (BC) patients and to investigate the association between the prevalence of PIK3CA H1047R mutation and clinical presentations. A proper blocker was designed in an allele-specific manner and optimized for PCR-based identification of the PIK3CA H1047R mutation. The established technique was validated in cell-free DNA samples from 196 recruited BC patients. The allele-specific PCR assay with a properly designed blocker was able to detect the H1047R mutant variant with 0.01%. By applying the newly established assay, 62 cases (31.6% of the total recruited cases) were found to carry a blood-circulating H1047R mutant. Wherein, the detected mutant rates increased with disease stages from 2/18 (11.1%) of stage I to 17/71 (23.9%) of stage II, 20/53 (37.7%) of stage III, and 23/31 (42.6%) of stage IV (p = 0.025), respectively. Higher frequencies of H1047R mutation were associated with late-stage (p = 0.033) or recurrence (p = 0.045) or metastatic patients (p = 0.049) as well as radiation-treated human epidermal growth factor receptor 2 (HER2) positive BC (p = 0.004). PIK3CA mutant carriers were frequently observed in patients under the age of 50 who had liver-metastasized or brain metastases or lymph node-invaded (p < 0.05). A novel allele-specific PCR assay with high sensitivity was established successfully for the detection of the PIK3CA H1047R mutation in clinical practice.

A novel PIK3CA hot-spot mutation in breast cancer patients detected by HRM-COLD-PCR analysis.

The PI3K protein is involved in the PI3K/AKT/mTOR pathway. Deregulation of this pathway through PIK3CA mutation is common in various tumors. The aim of this work is to identify hotspot mutation at exons 9 and 20 in Tunisian patients with sporadic or hereditary breast cancer. Hotspot mutations in exon 9 and exon 20 of the PIK3CA gene were identified by QPCR-High Resolution Melting followed by COLD-PCR and sequencing in 63 (42 sporadic cases and 21 hereditary cases) tumor tissues collected from Tunisian patient with breast cancer. MCF7, and BT20 breast cancer cell lines harboring the PIK3CA hotspot mutations E545K and H1047R in exon 9 and exon 20 respectively, were used as controls in HRM experiments. PIK3CA hotspot mutations were detected in 66.7% (28 out of 42) of sporadic BC cases, and in 14.3% (3 out of 21) of hereditary BC. The E545K and the H1048Y were the most prevalent mutations identified in patients with sporadic and hereditary BC, whereas the H1047R hotspot mutation was not found in our patients. Statistical analysis showed that PIK3CA mutation associated with an aggressive behavior in patients with sporadic BC, while it's correlated with age, tumor stage and tumor size in the group patients with hereditary breast cancer. Our results showed a novel PIK3CA hotspot mutation in Tunisian breast cancer patients detected by HRM-COLD-PCR. Moreover, the absence of PIK3CA hotspot mutation associated with good prognosis.

CarcSeq detection of lorcaserin-induced clonal expansion of Pik3ca H1047R mutants in rat mammary tissue.

Lorcaserin is a 5-hydroxytryptamine 2C (serotonin) receptor agonist and a nongenotoxic rat carcinogen, which induced mammary tumors in male and female rats in a 2-yr bioassay. Female Sprague Dawley rats were treated by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating bioassay dosing but for shorter duration, 12 or 24 wk. To characterize exposure and eliminate possible confounding by a potentially genotoxic degradation product, lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions, terminal plasma, mammary, and liver samples using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry. N-nitroso-lorcaserin was not detected, supporting lorcaserin classification as nongenotoxic carcinogen. Mammary DNA samples (n = 6/dose/timepoint) were used to synthesize PCR products from gene segments encompassing hotspot cancer driver mutations, namely regions of Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Tp53. Mutant fractions (MFs) in the amplicons were quantified by CarcSeq, an error-corrected next-generation sequencing approach. Considering all recovered mutants, no significant differences between lorcaserin dose groups were observed. However, significant dose-responsive increases in Pik3ca H1047R mutation were observed at both timepoints (ANOVA, P < 0.05), with greater numbers of mutants and mutants with greater MFs observed at 24 wk as compared with 12 wk. These observations suggest lorcaserin promotes outgrowth of spontaneously occurring Pik3ca H1047R mutant clones leading to mammary carcinogenesis. Importantly, this work reports approaches to analyze clonal expansion and demonstrates CarcSeq detection of the carcinogenic impact (selective Pik3ca H0147R mutant expansion) of a nongenotoxic carcinogen using a treatment duration as short as 3 months.

Unraveling the synergistic mechanisms of pyrotinib in combination with trastuzumab: Insights from the PHILA trial.

e13015 Background: There are many options for the treatment of advanced HER2-positive breast cancer. The results of PHILA study suggest that pyrotinib, trastuzumab and docetaxel may be considered as a potential first line treatment for patients with untreated HER2-positive metastatic breast cancer. We aimed to investigate the synergistic mechanisms of pyrotinib plus trastuzumab and compare them to trastuzumab plus pertuzumab, providing further insights for the PHILA trial. Methods: The efficacy of combination treatments was detected by cell vitality assay, Western blot and immunohistochemistry. The relevant mechanism was examined by flow cytometry and co-immunoprecipitation. Cell-derived xenografts and TUBO tumor model were utilized to evaluate the anti-tumor activity of rational combinations. Next-generation sequencing of circulating tumor DNA from a cohort of 18 patients in the PHILA trial was performed to evaluate molecular alterations associated with clinical response. Results: We demonstrated that pyrotinib plus trastuzumab superiorly inhibited cell growth than trastuzumab plus pertuzumab in models of HER2-dependent breast cancer. In addition to inhibiting HER family phosphorylation, pyrotinib plus trastuzumab synergistically reduced membrane HER2 levels by inducing receptor ubiquitination and internalization via HSP90 dissociation, which resulted in a comprehensive blockade of HER signaling pathway. Moreover, TUBO tumor model was utilized to further confirm the synergistic effects of pyrotinib and anti-neu antibody on HER2 signaling cascade. In both treatment groups of PHILA study, patients with aberrant mutations in HER2 signaling cascade showed a significantly shorter median progression-free survival compared to individuals with wild-type pathway. Correspondingly, in models of HCC1954 breast cancer cell harboring PIK3CA H1047R mutation, we did not observe a synergy between pyrotinib and trastuzumab. Conclusions: Pyrotinib and trastuzumab synergistically achieve the comprehensive blockade of HER signaling pathway in HER2-dependent breast cancer. Patients with advanced HER2-positive breast cancer, who are addicted to HER2 signaling cascade, could be considered for pyrotinib in combination with trastuzumab and docetaxel as an alternative first line treatment.

Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations

BackgroundRecent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype.ResultsIn this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases.ConclusionsUsing our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

Abstract A010: Cancer associated fibroblast dependency in a novel immunocompetent mouse model of bladder cancer

Abstract Introduction: The relationship between genotype and phenotype in targeted therapy has proven to be crucial for the successful use of most kinase inhibitors. In bladder cancer, the PI3K/AKT/mTOR pathway is widely activated by mutation but untargeted. Our study focuses on developing an immunocompetent, CRISPR-edited bladder cancer mouse model that mirrors common PI3K/AKT/mTOR pathway mutations. We established effective genotype-kinase inhibitor pairings using CRISPR-edited organoids and cancer associated fibroblasts (CAFs) to create a pro-tumorigenic environment in vivo. Methods and Results: Urothelial cells were isolated from the bladder of Rosa26 Lox-Stop-Lox-Cas9/GFP transgenic mice and grown as organoids in matrigel. These organoids were co-edited for Trp53 and Rb1 KO, and further editing of the Pten gene resulted in a nine-fold increase in sensitivity to the kinase inhibitor ipatasertib. Despite successful initial engraftment into mouse bladder, these edited cells regressed before tumor formation, and subcutaneous injection did not yield subcutaneous tumors. An alternate model featuring a 9p21 deletion with Trp53 KO also displayed similar regression post-engraftment into bladder or subcutaneous injections. However, co-injection with a previously established CAF line subcutaneously yielded palpable subcutaneous tumors that enlarged for 12 weeks. We then established a BBN bladder-tumor-derived CAF line, leading to the formation of tumors with squamous differentiation upon intramural co-injection with edited organoids. These models displayed immune-excluded architecture and a predominance of M2 over M1 macrophages. We also aimed to enhance the model's adaptability by introducing common PI3K/AKT/mTOR mutations, specifically TSC1/TSC2 KO, and Pik3ca hotspots, into our 9p21/Trp53 KO model. While TSC1/TSC2 KO lines exhibited elevated P-S6K signaling, they displayed resistance to Torin 1, Voxtalisib, and Everolimus—kinase inhibitors targeting mTORC1/2. In parallel, we employed CRISPR-mediated homologous recombination to successfully introduce a Pik3ca H1047R hotspot into the 9p21/Trp53 KO genotype, as evidenced by increased Phosphorylation of AKT compared to wild-type 9p21/Trp53 KO cell lines. This genotype resulted in approximately 80-fold selective increased sensitivity to pictilisib, a PI3K inhibitor compared to Pik3ca WT control. Conclusions: In this model, CAFs were required for successful engraftment of tumors into the bladder wall or subcutaneously. BUTU is a customizable model of bladder cancer and is suitable for direct targeting by kinase inhibitors while providing a platform for understanding the genotype-phenotype relationship in bladder cancer. We hope to use it as a tool to develop preclinical evidence on which to base precision medicine clinical trials. Citation Format: Philip H. Abbosh, Henkel Valentine. Cancer associated fibroblast dependency in a novel immunocompetent mouse model of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A010.

Abstract PO2-04-09: Combination of the PLK1 Inhibitor Onvansertib and the PI3Kα Inhibitor Alpelisib Overcomes Palbociclib Resistance in PIK3CA-mutated HR+ Breast Cancer

Abstract In the 1st line setting of hormone receptor-positive HER2 negative (HR+/HER2-) metastatic breast cancer, cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors (palbociclib, abemaciclib and ribociclib) are typically used in combination with endocrine therapy. Though initially effective, acquired resistance to either CDK4/6 inhibitors or endocrine therapy eventually leads to disease relapse in most patients. Activating mutations in PIK3CA, the gene encoding alpha catalytic subunit of phosphatidylinositol-4,5-bisphosphat 3-kinase (PI3K) are found in approximately 40% of HR+ breast cancer patients and have been implicated in mediating resistance to CDK4/6 inhibitors. The PI3Kα inhibitor alpelisib is currently approved for PI3KCA-mutant HR+ breast cancer after progression on CDK4/6 inhibitors. However, drug-associated resistance mechanisms may limit its clinical benefit. Synergistic combination therapies have the potential to improve efficacy and overcome resistance mechanisms. Polo-like kinase 1 (PLK1) is a serine-threonine-protein kinase, key cell cycle regulator that controls G2/M entry and mitotic progression, and that has been shown to mediate resistance to palbociclib in HR+ breast cancer. Onvansertib is an oral and highly specific PLK1 inhibitor currently on clinical development in solid tumors and hematological malignancies. Here we evaluated the potential of onvansertib to increase the activity of alpelisib in PIK3CA-mutant HR+ breast cancer preclinical models. The combination of onvansertib and alpelisib synergistically inhibited cell viability and/or colony formation in three PI3KCA-mutant HR+ breast cancer cell lines, MCF-7 (PIK3CA E545K), EFM-19 (PIK3CA H1047L), and T-47D (PIK3CA 1047R). A significant increase in apoptosis was observed in cells treated with the combination compared to either agent alone. We next tested the combination of onvansertib and alpelisib in patient-derived xenograft (PDX) models. For this purpose, palbociclib-resistant PIK3CA-mutant HR+ breast cancer PDXs were established from primary breast tumor (PDX HBCx-86, PIK3CA E545K) or from metastatic bone biopsies of patients who had progressed on endocrine therapy plus palbociclib (HBCx-180, PIK3CA H1047R) or on PI3Kα inhibitor (HBCx-134palboR31, PIK3CA H1047R). PDX tumors were grafted subcutaneously in nude mice and mice were treated with vehicle, onvansertib (45 mg/kg, oral, 5 days a week), alpelisib (25mg/kg, oral, 5 days a week) or the combination. The combination was well tolerated and showed superior anti-tumor activity than the single agents in the 3 PDX models. In the HBCx-86 PDX, although none of the single agents showed activity, the combination induced potent tumor growth inhibition. In the metastasis-derived PDX HBCx-134palboR31, the combination treatment induced pronounced tumor regression in 62% of mice (5/8), with complete response in 25% (2/8), while mice treated with the single agents showed tumor progression. Finally, in the HBCx-180 PDX, established from a patient with primary resistance to palbociclib, the efficacy of the combination was greater and more durable, with significant survival advantage, compared to the monotherapies. Collectively, our preclinical findings suggest that co-targeting PLK1 and PI3Kα with onvansertib and alpelisib respectively, may constitute a promising therapeutic combination strategy for patients with PIK3CA-mutant HR+ breast cancer failing to respond to first-line standard of care therapies. Citation Format: Sreeja Sreekumar, Pierre Painsec, Davis Klein, Dalia Gonzalez, Laura Sourd, Elodie Montaudon, Tod Smeal, Elisabetta Marangoni, Maya Ridinger. Combination of the PLK1 Inhibitor Onvansertib and the PI3Kα Inhibitor Alpelisib Overcomes Palbociclib Resistance in PIK3CA-mutated HR+ Breast Cancer [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 PO2-04-09.

Abstract PO4-16-05: Identification of functional HLA-A*11:01-restricted driver gene PIK3CA mutation specific T-cell receptors

Abstract The adoptive transfer of genetically engineered T-cell receptors (TCR-T) has emerged as a promising therapeutic strategy for the treatment of solid tumors. Recent clinical trials have demonstrated the safety and efficacy of TCR-T cell therapies against certain types of metastatic solid cancers. In our study, we aimed to investigate the potential of TCR-T cell therapies targeting neoantigens, which are exclusively expressed in cancer cells, with a focus on metastatic breast cancer (MBC). Specifically, we identified four driver missense mutations (PIK3CA E542K, E545K, H1047L, and H1047R) frequently observed in MBC patients and utilized computational tools to predict the generation of neoantigens. Utilizing NetMHCpan V.4.1, we performed screening of peptides spanning the mutation region and predicted a neoantigen epitope, PIK3CA_H1047L, to bind with HLA-A*11:01. To validate the presentation and stability of the neoantigen epitope, we employed TAP1-deficient K562 cells and observed stable expression of the mutant peptide-major histocompatibility complex (MHC) complex on the cell membrane. Subsequently, we isolated T cells from healthy donors possessing the HLA-A11:01 genotype, and subjected them to successive stimulations with neopeptide-pulsed dendritic cells. T cells stimulated with neopeptide were examined for neoantigen specificity using an IFN-γ ELISpot assay. Employing pMHC tetramer staining and single-cell sorting via flow cytometry, we successfully isolated five HLA-A11:01-restricted PIK3CA_H1047L-specific TCRs. Through lentivirus transduction of these TCRs into T cells, we achieved the generation of TCR-T cells that exhibited potent and specific activity exclusively against the PIK3CA_H1047L neoantigen. The functionality of the TCR-T cells was confirmed through assessments of CD137 expression, IFN-γ secretion, and cytotoxicity assays. In conclusion, our study demonstrates T cell responses towards neoantigens derived from PIK3CA, a commonly observed driver mutation in MBC. Furthermore, we successfully isolated five distinct neoantigen-specific TCRs that specifically recognized the PIK3CA_H1047L mutation and were restricted to the prevalent HLA-A*11:01 allele. The transfer of these TCRs into T cells resulted in the generation of TCR-T products exhibiting remarkable activity against the PIK3CA_H1047L neoantigen. These findings strongly encourage further investigation into TCRs targeting driver mutations in MBC, with the aim of advancing neoantigen-targeted TCR-T therapies into clinical trials. Additionally, the PIK3CA_H1047L neoantigen holds great promise as a potential candidate for the development of effective cancer vaccines. Citation Format: Meiying Shen, Xiaojian Han, Siyin Chen, Aishun Jin, Shengchun Liu. Identification of functional HLA-A*11:01-restricted driver gene PIK3CA mutation specific T-cell receptors [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 PO4-16-05.

Prevalence and effect of PIK3CA H1047R somatic mutation among Indian head and neck cancer patients

PIK3CA is one among the several mutated genes in cancer, including head and neck squamous cell carcinoma (HNSCC). H1047R is a hotspot somatic mutation in PIK3CA that occurs most frequently in several forms of cancers. Distribution of PIK3CA H1047R mutation in Indian HNSCC patients was screened and its effect on disease progression and response to treatment was analysed in this study. Genomic DNA was extracted from tumour biopsies of HNSCC patients (n = 48) and polymerase chain reaction coupled restriction fragment length polymorphism (PCR-RFLP) technique was used to screen for the mutation. Overall survival (OS) and Progression-free survival (PFS) of the patients were calculated in order to study effect of this mutation on survival and response to treatment respectively. Results showed that irrespective of patients’ criteria, twenty-five patients (52 %) carried a heterozygous form of mutation (His/Arg) and the rest (48 %) were wild type (His/His). The mean OS of the cohort with the mutation was 20.451 months (SE ± 1.710 months) while 26.31 months (SE ± 2.431) was in wild type population. PFS of the patients with the mutation was 18.612 months (SE ± 2.072), and for the wild type population, it was 26.31 months (SE ± 2.431). These observations suggest that Indian HNSCC patients with PIK3CA H1047R mutation have poor prognosis.

Different Oncogenes and Reproductive Histories Shape the Progression of Distinct Premalignant Clones in Multistage Mouse Breast Cancer Models

A remote carcinogen exposure can predispose to breast cancer onset decades later, suggesting carcinogen-induced mutations generate long-lived premalignant clones. How subsequent events influence the progression of specific premalignant clones remains poorly understood. Here, we generated multistage mouse models of mammary carcinogenesis by combining chemical carcinogen exposure (using 7,12-dimethylbenzanthracene, DMBA) with transgenes that enable inducible expression of one of two clinically relevant mammary oncogenes: c-MYC (MYC) or PIK3CAH1047R (PIK). In prior work, DMBA exposure generated mammary clones bearing signature HrasQ61L mutations, which only progressed to mammary cancer after inducible Wnt1 oncogene expression. Here, after an identical DMBA exposure, MYC versus PIK drove cancer progression from mammary clones bearing mutations in distinct Ras family paralogs. For example, MYC drove cancer progression from either Kras- or Nras-mutant clones, whereas PIK transformed Kras-mutant clones only. These Ras mutation patterns were maintained whether oncogenic transgenes were induced within days of DMBA exposure or months later. Completing a full-term pregnancy (parity) failed to protect against either MYC- or PIK-driven tumor progression. Instead, a postpartum increase in mammary tumor predisposition was observed in the context of PIK-driven progression. However, parity decreased the overall prevalence of tumors bearing Krasmut, and the magnitude of this decrease depended on both the number and timing of pregnancies. These multistage models may be useful for elucidating biological features of premalignant mammary neoplasia.

Abstract 1436: Cancer associated fibroblast dependency in a model of bladder cancer

Abstract Introduction: The relationship between genotype and phenotype in targeted therapy has proven to be crucial for most kinase inhibitors. In bladder cancer, the PI3K/AKT/mTOR pathway is widely activated by mutation but untargeted. Our study focuses on developing an immunocompetent, CRISPR-edited bladder cancer mouse model that mirrors common PI3K/AKT/mTOR mutations. We aim to establish genotype-effective kinase inhibitor pairings using in vitro CRISPR-edited organoids and Cancer Associated Fibroblasts (CAFs) to create a pro-tumorigenic environment in vivo. Methods and Results: Urothelial cells were isolated from the bladder of Rosa26 Lox-Stop-Lox-Cas9/GFP transgenic mice to develop organoids. These organoids were co-edited for Trp53 and Rb1 KO, and further editing of the Pten gene resulted in a nine-fold increase in sensitivity to the kinase inhibitor ipatasertib. Despite successful engraftment into mouse bladder, these edited cells regressed before tumor formation, and intradermal injection did not yield subcutaneous tumors. An alternate model featuring a 9p21 deletion with Trp53 KO also displayed similar regression post-engraftment into bladder or skin. However, co-injection with skin-cancer-derived CAFs subcutaneously yielded palpable subcutaneous tumors that grew for 12 weeks. Enhancing orthotopic model fidelity, we established a BBN bladder-tumor-derived CAF line, leading to the formation of tumors with squamous differentiation upon intramural co-injection with edited organoids. These models displayed immune-excluded architecture and a predominance of M2 over M1 macrophages. Following our accomplishments in establishing bladder tumors, we aimed to enhance our model's adaptability by introducing common PI3K/AKT/mTOR mutations, specifically TSC1/TSC2 KO and Pik3ca hotspots, into our 9p21/Trp53 KO model. While TSC1/TSC2 KO lines exhibited elevated P-S6K signaling, they displayed resistance to Torin 1, Voxtalisib, and Everolimus—kinase inhibitors targeting mTORC1/2. In parallel, we employed CRISPR-mediated HDR to successfully introduce a Pik3ca H1047R hotspot into our 9p21/Trp53 KO model, as evidenced by increased Phosphorylation of AKT compared to wild-type 9p21/Trp53 KO cell lines. Conclusions: Our research establishes the crucial role of CAFs in bladder tumor formation. Our now established customizable model of bladder cancer is suitable for direct targeting by kinase inhibitors while providing a platform for understanding the genotype-phenotype relationship in bladder cancer. Citation Format: Henkel Valentine, Uttam Satyal, Laura Bukavina, Hassan Uddin, Yousttena Beyamin, Rhea Arya, Philip Abbosh. Cancer associated fibroblast dependency in a model of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1436.

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 5768: Proximity labeling with molecular signal sensing of oncogenic PIK3CA

Abstract PIK3CA mutations are major oncogenic drivers for oncogenesis. Mutant PIK3CA continuously activates the PI3K pathway, transmitting oncogenic signals within the transformed cells. Despite decades of efforts focused on targeting oncogenic PIK3CA for cancer therapy, the precise molecular and functional machinery remains incomplete due to the molecular complexity of the PI3K pathway. Mapping of proximity proteins in the oncogenic PI3K pathway and identifying mutant signals will signify a deep understanding of oncogenesis and a broad range of therapeutic applications. In this study, we hypothesize that PIK3CA mutant cancer cells establish a unique molecular program networking with distinct proteins, which differs from that of cancer cells with wild-type (WT) PIK3CA. To test this hypothesis, we have developed a novel bioengineering approach to decode protein-protein interactions of oncogenic PIK3CA and identify key molecular partners, potentially providing therapeutic vulnerabilities in cancer cells. First, we applied the proximity labeling technique, based on the fusion of a promiscuous protein ligase to a targeting protein of interest (POI), featuring proximity-dependent biotinylation of interacting and neighboring proteins. The promiscuous biotin ligases, BioID and BioID2, were utilized to integrate WT PIK3CA or gain-of-function hotspot mutations in at E545K or H1047R residues. To optimize the labeling protocol and biotinylation conditions, the BioID and BioID2 plasmids were fused with each POI, incorporating multiple combinations of protein tags, including myc, HA, and red fluorescence protein. Lamin A was used as the positive control, and a total of 15 plasmids were constructed in pcDNA3.1 mammalian expression vector. The sizes of the plasmids and the gene sequences were validated by PCR amplification and Sanger sequencing. Next, we employed a programmable nuclease, the prokaryotic Argonaute (pAgo), assisted by fluorescence resonance energy transfer technology, to detect oncogenic signals derived from mutant PIK3CA in cell-free conditions. To test our methods, we engineered two cancer cell lines (DHSA-1426 and COSB) derived from naturally occurring hemangiosarcoma in dogs, to induce PIK3CA H1047R mutations using CRISPR/Cas9. Our technical approach enables temporal and spatial molecular sensing of oncogenic PIK3CA and its functional interactome in living cancer cells by integrating high-resolution molecular mapping with a pAgo-based detection system. Our ongoing work involves the standardization and optimization of proximity labeling for oncogenic PIK3CA across ontogenetically distinct cancer cells. Furthermore, we will determine the sensitivity and specificity of the integrated approach to achieve proximity labeling with programmable detection of molecular signals from oncogenic PIK3CA. Citation Format: Rong Li, Donghee Lee, Md Abdullah, Jong Hyuk Kim. Proximity labeling with molecular signal sensing of oncogenic PIK3CA [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 5768.

Abstract 1111: Poor radiation sensitivity and potential radiosensitizers for betel-nuts related head and neck squamous cell carcinoma in Taiwan

Abstract Background: Betel nut chewing might contribute to strong invasion &amp; treatment refractoriness in HNSCC from Taiwan. Betel-nuts exposed HNSCC cell line, TW2.6, had high PDL1, defective p53 mutation, p16 loss, &amp; BCL2 overexpression. In our studies, PI3K/AKT/mTOR inhibitors, anti-angiogenesis therapies, CDK4/6 inhibitors, DDR interventions, epigenetic modifications, and immunotherapy-containing regimens will be future combination options. The genomic signature of TW2.6 has been figured out in our study(AACR2020 &amp; 2023), too, mainly with PIK3CA H1047R mutation, high TMB(8.42 muts/Mb)/MSS, VEGF-A amplification, p53/MYC/HRAS/DDR2/PDGFRbeta/EPHB1/ATM mutations, FAT1 loss, amplification of TERT/FGF10/CCND3/SOX9/IL-7R/SDHA/RICTOR/FLCN, CDK12/PTPRD loss of function, CDK8 mutation, &amp; deletions of STK11/ARID1B/MITF/TNFAIP3 and molecularly-guided novel interventions will be designed. Purpose &amp; Methods: Polo-like kinase 1 inhibitor(volasertib), gluconate(blocking citrate transport &amp; glucose metabolism), IAP inhibitor(Debio1143), glutaminase 1 inhibitor(CB839) were tested on TW2.6 to evaluate synergistic effects with radiation. Results: TW2.6 was resistant to volasertib, gluconate, or radiation alone; but sensitive to Debio1143 and CB839. Volasertib significantly enhanced radiation. The flow cytometry revealed volasertib and radiation resulted in a significant G2/M arrest. Western blotting showed volasertib and radiation increased p-histone3, p-CDK1, &amp; cyclin B levels and induced apoptosis. Gluconate, CB839, or Debio1143 each had significant synergistic effects with radiation. The radiosensitization efficacy would be volasertib&amp;gt;gluconate&amp;gt;CB839&amp;gt;Debio1143. Conclusions: In Taiwan, patients with R/M HNSCC progressing within 3 months after CCRT could be rescued by early ICIs. Recently, sequential CCRT followed by ICI or Debio-1143 with CCRT showed potential survival benefits. In our studies, novel agents such as PLK inhibitor, gluconate, and CB839 had even better radiosensitization compared with Debio1143. Sequential CCRT with novel agents followed by ICI will be the future trend, esp. in biology-aggressive betel-nuts related locally advanced HNSCC in Taiwan. Citation Format: Jo-Pai Chen, Jui-Ying Chang, Ruey-Long Hong. Poor radiation sensitivity and potential radiosensitizers for betel-nuts related head and neck squamous cell carcinoma in Taiwan [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 1111.

Abstract A014: Oncogenic PIK3CA on therapeutic resistance and cellular heterogeneity in malignant vascular cancer

Abstract Angiosarcoma is an extremely rare and aggressive cancer that originates from endothelial cells lining blood or lymphatic vessels. The PI3K-AKT pathway plays a crucial role in angiogenesis and pathogenesis of this vascular cancer. Multi-omics studies have identified oncogenic mutations in the PIK3CA, PIK3R1, and PTEN genes involved in the PI3K-AKT pathway in human and canine angiosarcomas. Recently, the FDA approved alpelisib, a PI3K-alpha specific inhibitor, for breast cancer patients with PIK3CA mutations. However, less than 30% of patients treated with alpelisib showed an overall response. Thus, understanding how PIK3CA mutant cells acquire therapeutic resistance is of high importance, yet it remains largely unexplored. In this study, we aimed to determine the molecular effects of PIK3CA mutations on therapeutic resistance against PI3K inhibitors in angiosarcomas. Specifically, PIK3CA H1047R mutations were induced in DHSA-1426 and COSB canine hemangiosarcoma cell lines using CRISPR/Cas9-mediated gene editing, and mutant clones were clonally selected. RNA-seq analysis from the PIK3CA mutant cells and 74 canine hemangiosarcoma tissue samples demonstrated that the activation of the oncogenic PI3K pathway increased cytokine and chemokine secretion, possibly contributing to an immunogenic and inflammatory tumor phenotype. Alpelisib completely abrogated AKT phosphorylation and decreased cytokine and chemokine expression in PIK3CA mutant cells. However, alpelisib showed only moderate potency in inhibiting cell proliferation of the mutant cells, demonstrating partial resistance. Subsequently, our single-cell RNA-seq analysis identified drug-resistant cell clusters within the total cell population. Marker gene analysis revealed that the resistant cell clusters upregulated genes associated with the G protein pathway (GNAI1, VAV3, and GNG11), adenylyl cyclase pathway (PDE4D and PDE7B), calcium signaling pathway (RYR3 and CAMK2D), TGF-beta pathway (TGFB2 and TGFBR2), and the anti-apoptotic gene BCL2. These pathways are all associated with RAS-MAPK signaling, and we confirmed enhanced phosphorylation of ERK upon alpelisib treatment through western blotting. Further, pseudo-time analysis using Monocle 3 indicated that the resistant cell clusters shared a similar differentiation time point with the sensitive cell clusters within the PIK3CA mutant population. Our single-cell trajectory data suggest that therapeutic resistance to alpelisib could be independent of differentiation level. Instead, it may involve an evolutionary program controlled by specific selective pressures between resistant and sensitive cells, potentially associated with immune regulatory mechanisms. Our ongoing work is evaluating whether a cellular autonomous program that regulates cytokine secretion induced by PIK3CA mutation contributes to therapeutic resistance in the tumor microenvironment through in vivo experiment. This study will provide insights into potential mechanisms governing the cellular evolution process of PIK3CA mutant cells, which promote drug resistance. Citation Format: Donghee Lee, Emma Kozurek, Md Abdullah, Rong Li, Joanne Kim, Erin B. Dickerson, Jong Hyuk Kim. Oncogenic PIK3CA on therapeutic resistance and cellular heterogeneity in malignant vascular cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A014.

MTOR inhibitor introduce disitamab vedotin (RC48-ADC) rechallenge microtubule-chemotherapy resistance in HER2-low MBC patients with PI3K mutation.

This study aimed to explore the efficacy and potential mechanisms of rechallenge therapy with microtubule-targeting agents (MTAs) in patients with HER2-low metastatic breast cancer (MBC). We performed a systematic review to investigate the rechallenge treatment concept in the field of HER2-low MBC treatment and utilized a series of cases identified in the literature to illustrate the concept. Here we reported two clinical cases of HER2-low MBC patients whose disease progressed after prior treatment with MTAs such as docetaxel and vincristine. When rechallenged with disitamab vedotin ((RC48-antibody-drug conjugate (ADC), a monomethyl auristatin (MMAE) MTA)), both patients achieved a partial response and the final progression-free survival (PFS) was 13.5 and 9 months, respectively. Genomic profiling detected a PIK3CA H1047R mutation in the patients. The patients were treated with everolimus before being rechallenged with RC48, which may lead to a better response. This study further summarizes and analyzes the potential mechanism of the PI3K-AKT signaling pathway in MTA resistance and reveals that the PIK3CA H1047R mutation may be a potential molecular marker for the efficacy prediction of mTOR inhibitors, providing new insights and potential therapeutic strategies for the application of MTAs to MBC patients.

Genetic analysis of oligo-recurrence breast cancer: correlation with clinical outcomes

BackgroundWe aimed to identify the relationship between the genomic characteristics and clinical outcomes of oligo-metastatic breast cancer.MethodsOligo-metastatic breast cancer diagnosed by pathology from January 2001 and August 2019 were reviewed and we matched the poly-metastatic patients based on the clinicopathological features of patients included. Clinicopathological values and data of genomic alterations were collected. Oligo-recurrence (oligo-R) was defined as a situation where disease progression occurred in less than 5 anatomical sites and other anatomic areas still suppressed by the ongoing therapy.ResultsA total of 26 breast cancer patients were enrolled in our study, including 14 patients with strict oligo-metastatic disease (oligo-R > 6 months) and 12 with simultaneous poly-metastatic disease. PIK3CA, TP53 and ERBB2 were the most common shared alterations identified in patients included. Based on the median time of oligo-R, we divided the patients with oligo-metastasis into longer oligo-R group (oligo-R > 31.04 months) and shorter oligo-R group (oligo-R ≤ 31.04 months). The analysis of PIK3CA mutation sites showed that H1047R mutation was closely associated with oligo-metastasis, rather than poly-metastasis. H1047R mutation also predicted a better prognosis (oligo-R > 31.04 months) in oligo-metastatic breast cancer. In addition, HER2 positive was more likely to be related to a good outcome in patients with oligo-metastasis.ConclusionsThrough the genetic analysis of samples from oligo-metastasis, we found the prognostic values of PIK3CA H1047R and HER2 in oligo- and poly-metastasis. We improved the stratification of prognosis and provided new insights for biological behaviors of oligo-metastatic breast cancer.

PIK3CA mutations in cutaneous squamous cell carcinoma.

Oncogenic PIK3CA mutation activates phosphoinositide 3-kinase (PI3K) enzyme, and PI3K-AKT signaling activation induces several growth-regulatory transcription factors. PIK3CA mutations have attracted attention as biomarker in clinical trials of various inhibitors including PI3K inhibitors. About 80% of PIK3CA mutations in human cancers are observed in 'hot spot' regions: exon 9 (E542K and E545K) and exon 20 (H1047R). There were few reports about clinical significance of PIK3CA mutations in cutaneous cell carcinoma (cSCC). Thus, we investigate the prevalence of three PIK3CA hot spot mutations in 143 cases with cSCC and evaluate the correlation between the presence of these mutations and clinical characteristics by using ddPCR. The frequency of each E542K, E545K and H1047R PIK3CA mutations was 1.4% (2/143), 2.8% (4/143), and 0.7% (1/143) respectively. No significant correlation was found between PIK3CA mutations and clinical characteristics. Although additional basic researches and clinical trials are necessary, various inhibitors may be effective therapeutics for PIK3CA mutation-positive cSCC. Our study revealed the prevalence of PIK3CA mutations in cSCC.