PTEN Loss Research Articles

Establishment of a trastuzumab-resistant extramammary Paget disease model: loss of PTEN as a potential mechanism.

Extramammary Paget disease (EMPD) is a rare, cutaneous intraepithelial adenocarcinoma typically treated with wide local excision. Unfortunately, a number of patients with metastases show poor responses to chemotherapy. While some studies have explored trastuzumab's effectiveness against EMPD positive for human epidermal growth factor receptor 2 (HER2), trastuzumab resistance (TR) may emerge after anti-HER2 therapy. In this study, we established TR EMPD patient-derived xenografts (PDX) that replicated the histological and HER2 expression traits of naive EMPD tumours. Cancer gene analyses revealed a loss of the PTEN gene in TR tumours, which was further confirmed by immunohistochemical staining and immunoblotting to test for protein expression levels. Reduced PTEN levels correlated with increased protein kinase B (Akt) phosphorylation and p27 downregulation, suggesting a potential mechanism for trastuzumab resistance in EMPD cells. In the trastuzumab-sensitive EMPD-PDX mouse model, PTEN inhibitors partially restored trastuzumab-mediated tumour regression. The TR EMPD-PDX responded favourably to targeted therapy (lapatinib, abemaciclib, palbociclib) and chemotherapy (eribulin, docetaxel, trastuzumab deruxtecan). This study demonstrates an innovative TR EMPD-PDX model and introduces promising antineoplastic effects with various treatments for TR EMPD tumours.

Multivariate analysis of metabolic state vulnerabilities across diverse cancer contexts reveals synthetically lethal associations.

Targeting the distinct metabolic needs of tumor cells has recently emerged as a promising strategy for cancer therapy. The heterogeneous, context-dependent nature of cancer cell metabolism, however, poses challenges in identifying effective therapeutic interventions. Here, we utilize various unsupervised and supervised multivariate modeling approaches to systematically pinpoint recurrent metabolic states within hundreds of cancer cell lines, elucidate their association with tumor lineage and growth environments, and uncover vulnerabilities linked to their metabolic states across diverse genetic and tissue contexts. We validate key findings via analysis of data from patient-derived tumors and pharmacological screens, and by performing new genetic and pharmacological experiments. Our analysis uncovers new synthetically lethal associations between the tumor metabolic state (e.g., oxidative phosphorylation), driver mutations (e.g., loss of tumor suppressor PTEN), and actionable biological targets (e.g., mitochondrial electron transport chain). Investigating the mechanisms underlying these relationships can inform the development of more precise and context-specific, metabolism-targeted cancer therapies.

Endometrioid ovarian carcinoma landscape: pathological and molecular characterization.

Endometrioid ovarian cancers (EOvC) are usually managed as serous tumors. In this study, we conducted a comprehensive molecular investigation to uncover the distinct biological characteristics of EOvC. This retrospective multicenter study involved patients from three European centers. We collected clinical data and formalin-fixed paraffin-embedded (FFPE) samples for analysis at the DNA level using panel-based next-generation sequencing and array-comparative genomic hybridization. Additionally, we examined mRNA expression using NanoString nCounter® and protein expression through tissue microarray. We compared EOvC with other ovarian subtypes and uterine endometrioid tumors. Furthermore, we assessed the impact of molecular alterations on patient outcomes, including progression-free survival (PFS) and overall survival (OS). Preliminary analysis of clinical data from 668 patients, including 86 (12.9%) EOvC, revealed more favorable prognosis for EOvC compared with serous ovarian carcinoma (5-year OS of 60% versus 45%; P = 0.001) driven by diagnosis at an earlier stage. Immunohistochemistry and copy number alteration (CNA) profiles of 43 cases with clinical data and FFPE samples available indicated that EOvC protein expression and CNA profiles were more similar to endometrioid endometrial tumors than to serous ovarian carcinomas. EOvC exhibited specific alterations, such as lower rates of PTEN loss, mutations in DNA repair genes, and P53 abnormalities. Survival analysis showed that patients with tumors harboring loss of PTEN expression had worse outcomes (median PFS 19.6 months vs. not reached; P = 0.034). Gene expression profile analysis confirmed that EOvC differed from serous tumors. However, comparison to other rare subtypes of ovarian cancer suggested that the EOvC transcriptomic profile was close to that of ovarian clear cell carcinoma. Downregulation of genes involved in the PI3K pathway and DNA methylation was observed in EOvC. In conclusion, EOvC represents a distinct biological entity and should be regarded as such in the development of specific clinical approaches.

Combination Therapy Approach to Overcome the Resistance to PI3K Pathway Inhibitors in Gynecological Cancers.

The PI3K signaling pathway plays an essential role in cancer cell proliferation and survival. PI3K pathway inhibitors are now FDA-approved as a single agent treatment or in combination for solid tumors such as renal cell carcinoma or breast cancer. However, despite the high prevalence of PI3K pathway alterations in gynecological cancers and promising preclinical activity in endometrial and ovarian cancer models, PI3K pathway inhibitors showed limited clinical activity in gynecological cancers. In this review, we provide an overview on resistance mechanisms against PI3K pathway inhibitors that limit their use in gynecological malignancies, including genetic alterations that reactivate the PI3K pathway such as PIK3CA mutations and PTEN loss, compensatory signaling pathway activation, and feedback loops causing the reactivation of the PI3K signaling pathway. We also discuss the successes and limitations of recent clinical trials aiming to address such resistance mechanisms through combination therapies.

PTEN inhibition enhances sensitivity of ovarian cancer cells to the poly (ADP-ribose) polymerase inhibitor by suppressing the MRE11-RAD50-NBN complex.

Poly (ADP-ribose) polymerase inhibitors (PARPis) can effectively treat ovarian cancer patients with defective homologous recombination (HR). Loss or dysfunction of PTEN, a typical tumour suppressor, impairs double-strand break (DSB) repair. Hence, we explored the possibility of inhibiting PTEN to induce HR deficiency (HRD) for PARPi application. Functional studies using PTEN inhibitor VO-OHpic and PARPi olaparib were performed to explore the molecular mechanisms in vitro and in vivo. In this study, the combination of VO-OHpic with olaparib exhibited synergistic inhibitory effects on ovarian cancer cells was demonstrated. Furthermore, VO-OHpic was shown to enhance DSBs by reducing nuclear expression of PTEN and inhibiting HR repair through the modulation of MRE11-RAD50-NBN (MRN) complex, critical for DSB repair. TCGA and GTEx analysis revealed a strong correlation between PTEN and MRN in ovarian cancer. Mechanistic studies indicated that VO-OHpic reduced expression of MRN, likely by decreasing PTEN/E2F1-mediated transcription. Moreover, PTEN-knockdown inhibited expression of MRN, increased sensitivities to olaparib, and induced DSBs. In vivo experiments showed that the combination of VO-OHpic with olaparib exhibited enhanced inhibitory effects on tumour growth. Collectively, this study highlights the potential of PTEN inhibitors in combination therapy with PARPis to create HRD for HRD-negative ovarian cancers.

Single nucleus transcriptomics, pharmacokinetics, and pharmacodynamics of combined CDK4/6 and mTOR inhibition in a phase 0/1 trial of recurrent high-grade glioma.

Outcomes for adult patients with a high-grade glioma continue to be dismal and new treatment paradigms are urgently needed. To optimize the opportunity for discovery, we performed a phase 0/1 dose-escalation clinical trial that investigated tumor pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics following combined ribociclib (CDK4/6 inhibitor) and everolimus (mTOR inhibitor) treatment in recurrent high-grade glioma. Patients with a recurrent high-grade glioma (n = 24) harboring 1) CDKN2A / B deletion or CDK4 / 6 amplification, 2) PTEN loss or PIK3CA mutations, and 3) wild-type retinoblastoma protein (Rb) were enrolled. Patients received neoadjuvant ribociclib and everolimus treatment and no dose-limiting toxicities were observed. The median unbound ribociclib concentrations in Gadolinium non-enhancing tumor regions were 170 nM (range, 65 - 1770 nM) and 634 nM (range, 68 - 2345 nM) in patients receiving 5 days treatment at the daily dose of 400 and 600 mg, respectively. Unbound everolimus concentrations were below the limit of detection (< 0.1 nM) in both enhancing and non-enhancing tumor regions at all dose levels. We identified a significant decrease in MIB1 positive cells suggesting ribociclib-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) based comparisons of 17 IDH-wild-type on-trial recurrences to 31 IDH-wild-type standard of care treated recurrences data demonstrated a significantly lower fraction of cycling and neural progenitor-like (NPC-like) malignant cell populations. We validated the CDK4/6 inhibitor-directed malignant cell state shifts using three patient-derived cell lines. The presented clinical trial highlights the value of integrating pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics to assess treatment effects in phase 0/1 surgical tissues, including malignant cell state shifts. ClinicalTrials.gov identifier: NCT03834740 .

Clinicopathologic and genomic characteristics of patients with advanced ovarian, breast, and prostate cancer treated with poly (ADP-ribose) polymerase inhibitors (PARPi) in a real-world setting.

3159 Background: PARPis induce synthetic lethality in tumors that have homologous repair deficiencies (HRD). Due to relative rarity of HRD-related genomic and germline alterations across solid malignancies, it remains unclear which patients (pts) may derive most benefit from PARPis across different tumor types. Methods: We analyzed clinicopathologic and genomic characteristics of pts with advanced ovarian (OC, n=33), prostate (PC, n=28) and breast cancer (BC, n=13) treated with PARPi monotherapy at our institution. Only pts with HRD-related pathogenic alterations detected via next-generation sequencing of tumor tissue, with or without genetic susceptibility testing, were included. Results: Forty-one (55%) pts had a confirmed germline HRD-related gene alteration, of which 33 had BRCA1/2 and 8 had non-BRCA1/2 gene alterations. Average age of pts with BC was 54 years, OC 62 years, and PC 68 years. Median number of prior lines of therapy in the advanced setting was 1 (1-4) for OC and BC (0-7) and 3 (1-6) for PC. Median time to progression (TTP) in pts with OC was 8.3 months, BC 4.9 months and PC 3.6 months, likely due to i) majority of pts with OC received maintenance PARPi following favorable response to first-line chemotherapy, ii) pts with PC were older, and iii) more pts with PC had somatic rather than germline HRD-related mutations. Pts with germline vs somatic HRD mutation had longer median TTP in PC (5.0 vs 3.3 months) and BC (6.1 vs 4.0 months), but the opposite was true for OC (7.9 vs 10.8 months). Pathogenic TP53 mutations were identified in all but one pt with OC (32/33), 10/13 pts with BC, and 6/28 PCs. [table] Average TP53gene variant allele fraction (VAF) was similar in OC and BC (49.8% and 51.1%, respectively) but significantly lower in PC (26.4%), suggesting that TP53 mutations in PC were subclonal. No specific TP53 VAF or HRD-to- TP53 VAF ratio cut-off was associated with TTP or overall survival in any of the 3 cancer types. In addition, 7/28 (25%) of pts with PC had PTEN loss associated with modified response to PARPis, whereas none of the pts with BC had PTEN loss and only 3/13 had alterations of the PTEN/PI3K/AKTpathway. Conclusions: Double-strand DNA breaks caused by synergistic effects of aberrant TP53 and HRD-related genes can possibly explain the more favorable response of pts with OC and BC to PARPi, compared to those with PC. Co-alterations in other signaling pathways, epigenetic factors and/or tumor microenvironment likely play a role in the degree of response to PARPis, rather than TP53 clonality alone. In the era of expansion of genomic testing and new indications for PARPi larger genomic studies are needed to identify biomarkers of response to PARPi beyond HRD. [Table: see text]

Neratinib, a pan ERBB/HER inhibitor, restores sensitivity of PTEN-null, BRAFV600E melanoma to BRAF/MEK inhibition

IntroductionApproximately 50% of melanomas harbor an activating BRAFV600E mutation. Standard of care involves a combination of inhibitors targeting mutant BRAF and MEK1/2, the substrate for BRAF in the MAPK pathway. PTEN loss-of-function mutations occur in ~40% of BRAFV600E melanomas, resulting in increased PI3K/AKT activity that enhances resistance to BRAF/MEK combination inhibitor therapy.MethodsTo compare the response of PTEN null to PTEN wild-type cells in an isogenic background, CRISPR/Cas9 was used to knock out PTEN in a melanoma cell line that harbors a BRAFV600E mutation. RNA sequencing, functional kinome analysis, and drug synergy screening were employed in the context of BRAF/MEK inhibition.ResultsRNA sequencing and functional kinome analysis revealed that the loss of PTEN led to an induction of FOXD3 and an increase in expression of the FOXD3 target gene, ERBB3/HER3. Inhibition of BRAF and MEK1/2 in PTEN null, BRAFV600E cells dramatically induced the expression of ERBB3/HER3 relative to wild-type cells. A synergy screen of epigenetic modifiers and kinase inhibitors in combination with BRAFi/MEKi revealed that the pan ERBB/HER inhibitor, neratinib, could reverse the resistance observed in PTEN null, BRAFV600E cells.ConclusionsThe findings indicate that PTEN null BRAFV600E melanoma exhibits increased reliance on ERBB/HER signaling when treated with clinically approved BRAFi/MEKi combinations. Future studies are warranted to test neratinib reversal of BRAFi/MEKi resistance in patient melanomas expressing ERBB3/HER3 in combination with its dimerization partner ERBB2/HER2.

Chemotherapy-induced PTEN-L secretion promotes the selection of PTEN-deficient tumor cells

BackgroundPTEN loss has been identified in various tumor types and is linked to unfavorable clinical outcomes. In addition to PTEN mutation, multiple mechanisms contribute to PTEN loss during tumor development. However, the natural selection process of PTEN-deficient tumor cells remains unclear. Here, we aimed at further elucidating the role of PTEN-L in tumor progression.MethodsPTEN knockout cell lines were generated using CRISPR/Cas9 technology. Ni-NTA affinity column chromatography was employed for PTEN-L purification. Tumor cell metastasis was evaluated in murine models and observed using the IVIS Spectrum Imaging System. RNA-sequencing, western blotting, PCR, flow cytometry, and cell proliferation assays were employed to investigate tumor cell dormancy and related mechanisms.ResultsThe chemotherapeutic drugs, cisplatin, paclitaxel, and doxorubicin, induced tumor cells to secrete PTEN-long (PTEN-L), which shields PTEN-deficient tumor cells from chemotherapy-induced apoptosis better than it shields PTEN-intact cells. Further investigation revealed that PTEN-L treatment induced dormancy in PTEN-null tumor cells, characterized by an increase in p16 and p27 levels, cell-cycle arrest, reduced cell proliferation, and enhanced DNA repair. Furthermore, PTEN-L treatment selectively promoted the accumulation and growth of PTEN-null tumor cells in the lungs of C57BL/6J mice, while evading immune surveillance. Mechanistically, PTEN-L induced dormancy in PTEN-null tumor cells by activating the p38 signaling pathway. Addition of a p38 inhibitor effectively reversed dormancy and growth of PTEN-deficient tumor cells in the lungs. We also demonstrated that PTEN expression played a pivotal role in determining the outcome of PTEN-L-mediated antitumor therapy.ConclusionsIn summary, PTEN-L was identified as a potent inducer of dormancy in PTEN-deficient tumor cells, which increased their efficient selection within the tumor microenvironment.

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 PO2-06-07: A Randomized Phase II Comparison of Single-Agent Carboplatin versus the Combination of Carboplatin and Everolimus for the Treatment of Advanced Triple Negative Breast Cancer

Abstract Background: Both TNBC and BRCA-1 associated breast cancers are sensitive to DNA cross-linking agents such as platinum compounds due to defective DNA repair by homologous recombination. TNBCs are also associated with a high frequency of PTEN loss, which can lead to activation of the mTOR pathway resulting in tumor cell growth and proliferation. mTOR activation can confer resistance to platinum agents, and this phenomenon may be reversible by the addition of an mTOR inhibitor, such as everolimus. A prior phase II single arm trial of carboplatin and everolimus in patients (pts) with advanced TNBC demonstrated good tolerability and preliminary efficacy. Methods: We conducted a phase II, multicenter, randomized trial in pts with advanced TNBC, who had received 0-3 prior lines of therapy. Pts were randomly assigned (in a 2:1 ratio) to carboplatin and everolimus or carboplatin alone. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), overall response rate (ORR), clinical benefit rate (CBR), and safety. We planned to enroll 72 pts which would provide &amp;gt; 80% power for a one-sided log-rank test at the 5% level of significance, to detect a 2.5 month improvement in median PFS between treatment groups, assuming a median PFS of 2.5 months in the control group. An interim analysis for futility was planned after 36 events with a stopping boundary of P &amp;lt;0.375 for the comparison of PFS, using an Obrien-Fleming spending function. Of note, the trial was stopped earlier than planned based on a sensitivity/tipping point analysis which indicated that terminating the trial 3 months ahead of schedule would have no substantial impact on the primary endpoint. Results: A total of 56 pts were randomized between 2015 and 2022, of whom 36 received carboplatin/everolimus and 20 carboplatin alone. The median age of the population was 62.8 years (range: 33-87) and about 20% of pts had BRCA-1 or BRCA-2 mutations. In the overall population, pts had received a median of 1 (range 1-3) prior line of therapy in the metastatic setting, with 75% of pts receiving prior chemotherapy and 47% prior carboplatin. The median PFS was significantly improved in pts who received carboplatin and everolimus (4.7 months) versus those who received carboplatin alone (2.1 months; HR: 0.37; 95% CI: 0.19-0.72; p=0.0042). Overall survival was 22.1 months in pts on the combination versus 14.4 months on carboplatin alone (HR: 0.73; 95% CI: 0.30-1.72; p=0.3106). CBR was 61% with the combination vs 50% with carboplatin monotherapy, as shown in Table 1. Most common adverse events (AEs) on carboplatin and everolimus included thrombocytopenia (81%), anemia (69%), leukopenia (67%), fluid retention (64%), and neutropenia (61%). Overall, Grade 3/4 events occurred in 75% of pts (83% of pts on combination vs 60% on carboplatin alone) and were primarily anemia (40% vs 17%), thrombocytopenia (47% vs 0%), and neutropenia (20% vs 0%). Of note, there was an increase in thrombocytopenia with the addition of everolimus (All Grades: 81% vs 35%, Grade 3/4: 39% vs 0%). Conclusions: Treatment options for TNBC are limited due to a lack of targeted therapies. The combination of carboplatin and everolimus in this study was associated with a 63% reduction in risk of progression or death in pts with metastatic TNBC. The regimen was well tolerated and provides a promising treatment option for pts with advanced TNBC. Table 1. Results in Patients on Carboplatin and Everolimus versus Carboplatin Alone NE= Not Estimable; 1Unevaluable patients treated as non-responders in CBR and ORR calculations. Citation Format: Rima Patel, Jami Fukui, Paula Klein, Erin Moshier, Charles Shapiro, Anupama Goel, Julie Fasano, Theresa Shao, Aarti Bhardwaj, Rita Vaccaro, Gargi Atul Joshi, Joseph Sparano, Amy Tiersten. A Randomized Phase II Comparison of Single-Agent Carboplatin versus the Combination of Carboplatin and Everolimus for the Treatment of Advanced Triple Negative 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 PO2-06-07.

Abstract PO4-14-03: Results of Comprehensive Genomic Profiling of Metastatic Breast Cancer Patients at a Single Institute in Japan

Abstract BACKGROUND. Comprehensive genomic profiling (CGP) was officially approved by the Japanese National Health Insurance System in June 2019. Currently, three CGP tests are available: FoundationOne CDx (F1CDx), OncoGuide NCC Oncopanel (NCC), and FoundationOne Liquid CDx (F1LCDx). This study examines the clinical significance of CGP testing in patients with metastatic breast cancer (mBC) in Japan. METHODS. The subjects were 105 patients (pts) who underwent CGP testing at our hospital from June 2019 to July 2023. Of these, 64 pts received tissue panels for F1CDx, 25 pts for F1LCDx, and 16 pts for others. Based on the reports from the testing companies and the expert panel committee's review, we evaluated the percentage of patients for whom matched therapy (MT) was recommended, whether MT was performed, and the prognosis of the group of patients for whom MT was performed. Overall survival was analyzed with the log-rank test. RESULTS. Of the 105 patients who underwent CGP testing, 47 pts (44.8%) were recommended MT corresponding to ESCAT(ESMO Scale for Clinical Actionability of molecular Targets) ranking I/II or clinical trials due to genetic alterations, and only 7 pts (6.7%) reached those treatments. Three HER2-negative patients were prescribed anti-HER2 therapy for ERBB2 amplification, one patient was prescribed entrectinib for NTRK fusion, and three patients participated in clinical trial for ERBB2 mutation and FGFR1 rearrangement. If ESCAT ranking beyond I/II and recommended therapy from FoundationOne report (e.g Abemaciclib for CCND1 amplification, Everolimus for PTEN loss)are added, 64 pts (60.1%) were considered these treatments, and 19 pts (18.1%) actually received these treatment. Among these 19 pts, overall survival was significant improved that of patientswho did not reach treatment (21.1 m vs 6.2 m, p=0.0097). CONCLUSIONS. We examined the rate of MT attainment by CGP in mBC at a single institute. We found that the rate of MT attainment with ESCAT ranking I/II, or clinical trials corresponding to genetic mutations, was low. The overall survival of patients who underwent MT, which can be performed by Japanese insurance, was significantly different from that of patients who did not undergo MT. Low accessibility of ESCAT ranking I/II MT may be because, in Japan, CGP testing is available only for patients who have completed or are expected to complete standard therapy. Early use of CGP testing and an increase in clinical trials will be desirable in Japan. Citation Format: Tomomi Kon, Hiroshi Tada, Minoru Miyashita, Akiko Ebata, Narumi Harada-Shoji, Yohei Hamanaka, Miku Sato, Mika Yanagaki, Satoko Tsunokake, Tokiwa Motonari, Asumi Yamazaki, Takanori Ishida. Results of Comprehensive Genomic Profiling of Metastatic Breast Cancer Patients at a Single Institute in Japan [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 PO4-14-03.

Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids

Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.

Combined deletion of MEN1, ATRX and PTEN triggers development of high-grade pancreatic neuroendocrine tumors in mice

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous group of tumors that exhibit an unpredictable and broad spectrum of clinical presentations and biological aggressiveness. Surgical resection is still the only curative therapeutic option for localized PanNET, but the majority of patients are diagnosed at an advanced and metastatic stage with limited therapeutic options. Key factors limiting the development of new therapeutics are the extensive heterogeneity of PanNETs and the lack of appropriate clinically relevant models. In that context, genomic sequencing of human PanNETs revealed recurrent mutations and structural alterations in several tumor suppressors. Here, we demonstrated that combined loss of MEN1, ATRX, and PTEN, tumor suppressors commonly mutated in human PanNETs, triggers the development of high-grade pancreatic neuroendocrine tumors in mice. Histopathological evaluation and gene expression analyses of the developed tumors confirm the presence of PanNET hallmarks and significant overlap in gene expression patterns found in human disease. Thus, we postulate that the presented novel genetically defined mouse model is the first clinically relevant immunocompetent high-grade PanNET mouse model.

Current Systemic Therapy in Men with Metastatic Castration-Sensitive Prostate Cancer.

This review aims to explore the evolving landscape of treatments available for metastatic castration-sensitive prostate cancer (mCSPC) patients. In less than a decade, evidence was chronologically provided that (1) systemic treatment intensification with docetaxel improves outcomes, including survival, in men with mCSPC, (2) then that these outcomes are also improved when a second-generation androgen receptor pathway inhibitor (ARPI) is combined with androgen deprivation therapy (ADT), and (3) using a "triplet systemic therapy," which consists in the combination of ADT, an ARPI and docetaxel, further improves outcomes, including survival. Radiotherapy to the prostate combined with ADT alone is now recommended in men with low-volume mCSPC. Combining prostate radiotherapy and intensified systemic treatment including abiraterone may be synergistic as suggested in the PEACE-1 trial. Also, the role of metastases-directed local therapies (mostly stereotactic radiotherapy) is currently being assessed in phase 3 trials. Finally, the integration of biomarkers (e.g. BRCA2 gene alterations, PTEN loss, PSMA expression) for decision making is not currently established, though trials are also currently underway. Importantly, most evidence currently available was obtained in men with de novo metastases, while for those with metastatic relapse after definitive local treatment, the role of treatment intensification is less well established. Treatment intensification is nowadays the standard of care for patients with de novo mCSPC as it leads to outcomes improvement, including survival, and the standard of care is evolving almost on a yearly basis.

Exploring The Prognostic Significance of SET-Domain Containing 2 (SETD2) Expression in Advanced and Castrate-Resistant Prostate Cancer.

SET-domain containing 2 (SETD2) is a histone methyltransferase and an epigenetic modifier with oncogenic functionality. In the current study, we investigated the potential prognostic role of SETD2 in prostate cancer. A cohort of 202 patients' samples was assembled on tissue microarrays (TMAs) containing incidental, advanced, and castrate-resistant CRPCa cases. Our data showed significant elevated SETD2 expression in advanced and castrate-resistant disease (CRPCa) compared to incidental cases (2.53 ± 0.58 and 2.21 ± 0.63 vs. 1.9 ± 0.68; p < 0.001, respectively). Interestingly, the mean intensity of SETD2 expression in deceased vs. alive patients was also significantly different (2.31 ± 0.66 vs. 2 ± 0.68; p = 0.003, respectively). Overall, high SETD2 expression was found to be considered high risk and was significantly associated with poor prognosis and worse overall survival (OS) (HR 1.80; 95% CI: 1.28-2.53, p = 0.001) and lower cause specific survival (CSS) (HR 3.14; 95% CI: 1.94-5.08, p < 0.0001). Moreover, combining high-intensity SETD2 with PTEN loss resulted in lower OS (HR 2.12; 95% CI: 1.22-3.69, p = 0.008) and unfavorable CSS (HR 3.74; 95% CI: 1.67-8.34, p = 0.001). Additionally, high SETD2 intensity with ERG positive expression showed worse prognosis for both OS (HR 1.99, 95% CI 0.87-4.59; p = 0.015) and CSS (HR 2.14, 95% CI 0.98-4.68, p = 0.058). We also investigated the protein expression database TCPA, and our results showed that high SETD2 expression is associated with a poor prognosis. Finally, we performed TCGA PRAD gene set enrichment analysis (GSEA) data for SETD2 overexpression, and our data revealed a potential association with pathways involved in tumor progression such as the AMPK signaling pathway, the cAMP signaling pathway, and the PI3K-Akt signaling pathway, which are potentially associated with tumor progression, chemoresistance, and a poor prognosis.

The Molecular Evolution of Melanoma Distant Metastases

The evolution of primary melanoma to lymph node and distant metastasis is incompletely understood. We examined the genomic diversity in melanoma progression in matched primary melanomas, lymph node and distant metastases from 17 patients. Fluorescence in situ hybridization (FISH) analysis revealed cancer cell fractions with monotonic copy number alterations (CNAs), including PHIP gain and PTEN loss, in the metastatic cascade. By contrast, the cancer cell fraction with CNAs for BPTF and MITF was reduced in lymph node metastases but increased in distant metastases. Separately, the cancer cell fraction with NCOA3 CNA was comparable in primary tumors and lymph nodes yet increased in distant metastases. These results suggest enrichment of the PI3K and MITF pathways in the transition through the metastatic cascade. By contrast, next-generation sequencing analysis did not identify a consistent pattern of changes in variant allele frequency (VAF) while revealing several intriguing findings, including decreased VAF in distant metastases and distinct drivers in lymph node versus distant metastases. These results provide evidence that distant melanoma metastasis does not always emanate from lymph node metastasis. These results enhance our understanding of clonal patterns of melanoma metastasis, with possible implications for targeted therapy and metastasis competency.

Abstract 3043: MYC activation and PTEN loss synergistically activate proliferation in a mouse model of lethal prostate cancer

Abstract Prostate cancer kills 30,000 men per year in the U.S. Yet, of all primary prostate cancers, only a relatively small percentage are aggressive and have the potential to become life threatening. In terms of genomic copy number alterations, concurrent amplification of MYC and loss of tumor suppressor PTEN is the strongest predictor of prostate cancer specific mortality. We have previously shown, in a novel mouse model, that combined MYC overexpression and Pten loss results in the development of precursor lesions (PIN) that lead to aggressive metastatic prostatic adenocarcinoma, yet overexpression of MYC alone or Pten loss alone result only in precursor lesion development. Here, we performed genome wide transcriptome analysis using bulk RNA seq to obtain clues regarding the synergy between MYC overexpression and Pten loss. Prostates harboring PIN lesion were dissected from mice engineered to have MYC activation alone, Pten loss alone, and the combination thereof (BMPC mice), as well as invasive primary adenocarcinomas and metastatic lesions from BMPC mice. Principal Components Analysis (PCA) analysis separated the lesions into highly distinct groups. Genes contributing the most to the first principal component were related to inflammation and cell proliferation/cell cycle progression by functional annotation. The proliferation-related genes were increased somewhat by MYC overexpression alone, were not increased Pten loss, and were more consistently increased by combined MYC overexpression and Pten loss in BMPC. Strikingly, mice with combined MYC overexpression and Pten loss showed approximately 2-4 fold higher Ki67 proliferation index than mice with MYC activation alone. PIN lesions driven by MYC overexpression or PTEN loss alone were characterized by Gene Set Enrichment Analysis (GSEA). Among the pathways with the highest negative enrichment scores in PIN samples with PTEN loss were those that generally describe ribosome biogenesis and translation. PIN lesions with Pten loss showed lower hybridization signals for the 45S rRNA, which was increased by MYC alone and somewhat more in BMPC PIN lesions. Our results demonstrate that combined activation of MYC and Pten loss drive increased cell proliferation beyond that seen with either gene alteration alone. These findings suggest that a key part of the molecular mechanisms by which MYC and PTEN alterations may synergize to drive aggressive prostate cancer is related to increased tumor cell proliferation. Surprisingly, we uncovered the novel finding that Pten loss, in the absence of MYC overexpression, is associated with decreased expression of genes coding for proteins that mediate translation, as well as decreased 45S pre-rRNA. Since ribosome biogenesis is a key driver of cell proliferation, it is likely that loss of both copies of PTEN alone, in the absence of MYC overexpression, would be selected against in early lesions in prostate cancer. Citation Format: Michael Rubenstein, Tracy Jones, Jessica Hicks, Apurv Rege, Qizhi Zheng, Carolina Gomes-Alexandre, Gretchen Hubbard, Srinivasan Yegnasubramanian, Charles Bieberich, Angelo DeMarzo. MYC activation and PTEN loss synergistically activate proliferation in a mouse model of lethal prostate 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 3043.

Abstract 6143: Association of polygenic risk score and prostate tumor biomarkers with lethal prostate cancer

Abstract Prostate cancer (PCa) is a highly heritable cancer. While polygenic risk scores (PRS) stratify overall PCa risk among cancer-free men, they have not been reported to predict lethal outcomes in PCa patients. We aimed to examine to what extent PRS modifies associations between tumor biomarkers with cancer prognosis after diagnosis. We included men with primary, non-metastatic PCa in the Health Professionals Follow-up Study (HPFS) and Physicians’ Health Study (PHS). The PRS was constructed with 451 risk variants identified from prior PCa GWAS, measured on DNA from blood or buccal, standardized by adjusting for mean and standard deviation (SD) based on men without PCa with array-based genotyping data in HPFS/PHS. Prostate tumor biomarkers of molecular subtypes (TMPRSS2:ERG fusion, PTEN loss, and TP53 missense mutations from validated immunohistochemistry assays), insulin/lipid signaling pathways (FASN, IR, and IGF1-R), and cellular proliferation and tumor microenvironment (Ki-67, apoptosis rates, and angiogenesis) were measured on tumor tissue from radical prostatectomy or TURMP. Sample sizes varied by tumor biomarkers, ranging from 214 for angiogenesis markers to 598 for ERG protein expression. We first used either logistic or negative binomial regression to evaluate the association between the PRS and the protein expression (levels) of each individual biomarker, and then evaluated the addition of multiplicative interaction terms between PRS and biomarkers to logistic regression models for lethal PCa, defined as metastases or death from PCa. Lastly, we evaluated the predictive performance for lethal PCa by area under the curve (AUC). All models adjusted for age at diagnosis and Gleason score. Among all patients included in the analysis, the median age at diagnosis was 66 (interquartile range: 62-70) years, with 20% having a Gleason score≥8 and 10% having lethal PCa. PRS showed no association with lethal PCa (OR per SD increase PRS=0.95, 95%CI 0.85-1.06) or most tumor biomarkers, except for the apoptosis index (per SD in PRS corresponding to 0.22 units increase, 95%CI 0.02-0.42). Statistically significant interactions were found between PRS and p53 overexpression (p interaction = 1.6x10-5), PTEN loss (p=3.7x10-3), FASN (p=0.03), and Ki-67 (p=1.6x10-3), suggesting that compared to men with a PRS higher than the median, TP53 missense mutation, PTEN loss, higher FASN intensity, and higher Ki-67 indices were more strongly associated with lethal PCa in patients with lower PRS. Incorporating PRS with tumor biomarkers into predictive models enhanced the AUC beyond a model with tumor biomarker and age. The largest improvements were observed when combining the PRS with Ki-67 and apoptosis, showing absolute increases of 5% and 7% respectively. In conclusion, PRS integration with tumor biomarkers at diagnosis potentially improves the prediction of lethal PCa, thereby informing more targeted management strategies. Citation Format: Anqi Wang, Anna Plym, Konrad H. Stopsack, Lorelei A. Mucci, Kathryn L. Penney. Association of polygenic risk score and prostate tumor biomarkers with lethal prostate 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 6143.

Abstract 1954: Development of highly potent, covalent, and selective inhibitors to target PI3Kα in cancer

Abstract Aberrant PI3K signaling is linked to various forms of cancer and proliferative disease. Approved clinical therapies targeting overactive PI3K are scarce and often accompanied by severe adverse effects due to lack of PI3K isoform selectivity. We recently reported a proprietary covalent proximity scanning (CoPS) approach to selectively target distant cysteines outside of the ATP-binding pocket in kinases. Using CoPS, we developed isoform specific, irreversible covalent PI3Kα inhibitors (cPI3Kαi) with outstanding target engagement profiles. Here, we report structure-guided scaffold and linker-optimizations resulting in cPI3Kαi with long-term and rapid inhibition of PI3Kα. Cellular activity was improved exploiting additional protein-inhibitor interactions inspired by X-ray crystallographic data, and introduction of structural elements to increase cell permeability. The cPI3Kαi series showed enhanced cellular potency and excellent on-target engagement. Kinetic parameters and covalent bond formation were determined using TR-FRET, nanoBRET tracer displacement assays, and LCMS/MS based proteomics. With our new cPI3Kαi, IC50s of PKB/Akt phosphorylation, pPKB/Akt recovery after drug washout, and growth inhibition experiments were performed in cancer cell lines harboring activating PIK3CA or inactivating PTEN mutations. Efficient and long-lasting inhibition of PKB/Akt phosphorylation was observed in PIK3CA mutant cell lines, while loss of PTEN was associated with partial responses, consistent with contributions of other class I PI3K isoforms to signal reactivation. A dynamic FOXO cytosolic/nuclear translocation assay was used to monitor distal PI3K signaling: cPI3Kαi blunted PI3K signaling rapidly with rates identical to the reversible BYL719/alpelisib. After drug washout, inhibition by BYL719/alpelisib was relieved within minutes, while a complete and prolonged shutdown of downstream signaling events was observed in PIK3CA mutant cell lines but not in cell lines where PTEN function was lost. This relapse in downstream signaling explains the lack of efficacy of PI3Kα selective inhibitors in models with PTEN loss and demonstrates that persistent inhibition across the PI3K pathway is required for efficacy. In summary, the new cPI3Kαi display outstanding specificity and cellular activity in PIK3CA-mutated cancer cells, and support the further development of cPI3Kαi as future clinical candidates and an alternative to established reversible inhibitors. Citation Format: Luka Raguž, Theodora A. Constantin, Lukas Bissegger, Erhan Keleş, Clara Orbegozo, Thorsten Schäfer, Rohitha Sriramaratnam, Chiara Borsari, Matthias P. Wymann. Development of highly potent, covalent, and selective inhibitors to target PI3Kα in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1954.