Phosphatidylinositol 3-kinase Pathway Research Articles

ABCG2 Mediates Resistance to the Dual EGFR and PI3K Inhibitor MTX-211 in Cancer Cells

MTX-211 is a first-in-class dual inhibitor of epidermal growth factor receptor (EGFR) and phosphoinositide-3 kinase (PI3K) signaling pathways with a compelling pharmaceutical profile and could enhance the effectiveness of mitogen-activated protein kinase kinase (MEK) inhibitor therapy in colorectal tumors with KRAS mutations. However, the specific mechanisms contributing to the acquired resistance to MTX-211 in human cancers remain elusive. Here, we discovered that the overexpression of the ATP-binding cassette (ABC) drug transporter ABCG2, a prevalent mechanism associated with multidrug resistance (MDR), could diminish the effectiveness of MTX-211 in human cancer cells. We showed that the drug efflux activity of ABCG2 substantially decreased the intracellular accumulation of MTX-211 in cancer cells. As a result, the cytotoxicity and effectiveness of MTX-211 in suppressing the activation of the EGFR and PI3K pathways were significantly attenuated in cancer cells overexpressing ABCG2. Moreover, the enhancement of the MTX-211-stimulated ATPase activity of ABCG2 and the computational molecular docking analysis illustrating the binding of MTX-211 to the substrate-binding sites of ABCG2 offered a further indication for the interaction between MTX-211 and ABCG2. In summary, our findings indicate that MTX-211 acts as a substrate for ABCG2, underscoring the involvement of ABCG2 in the emergence of resistance to MTX-211. This finding carries clinical implications and merits further exploration.

Evaluation of copanlisib in combination with eribulin in triple negative breast cancer patient-derived xenograft models.

The phosphoinositide 3-kinase (PI3K) pathway regulates essential cellular functions and promotes chemotherapy resistance. Activation of PI3K pathway signaling is commonly observed in triple negative breast cancer (TNBC). However previous studies that combined PI3K pathway inhibitors with taxane regimens have yielded inconsistent results. We therefore set out to examine whether the combination of copanlisib, a clinical grade pan-PI3K inhibitor, and eribulin, an antimitotic chemotherapy approved for taxane-resistant metastatic breast cancer, improves the anti-tumor effect in TNBC. A panel of 8 TNBC patient-derived xenograft (PDX) models were tested for tumor growth response to copanlisib and eribulin, alone or in combination. Treatment induced signaling changes were examined by reverse phase protein array, immunohistochemistry, and 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET). Compared to each drug alone, the combination of eribulin and copanlisib led to enhanced tumor growth inhibition, which was observed in both eribulin-sensitive and -resistant TNBC PDX models, regardless of PI3K pathway alterations or PTEN status. Copanlisib reduced PI3K signaling and enhanced eribulin induced mitotic arrest. The combination enhanced induction of apoptosis compared to each drug alone. Interestingly, eribulin upregulated PI3K pathway signaling in PDX tumors, as demonstrated by increased tracer uptake by 18F-FDG PET scan of PDX tumor and AKT phosphorylation by immunohistochemistry. These changes were inhibited by the addition of copanlisib. These data support further clinical development for the combination of copanlisib and eribulin. These data led to a phase I/II trial of copanlisib and eribulin in patients with metastatic TNBC.

Abstract PO4-26-06: Identifying mutation-independent signaling pathway activation in molecular subtypes of triple negative breast cancers

Abstract Introduction Triple negative breast cancer (TNBC) is an aggressive cancer defined by the absence of estrogen receptors (ER), progesterone receptors (PR), and lack of human epidermal growth factor receptor 2 (HER-2). We previously identified molecular subgroups with distinct mutational, epigenetic, and clinicopathologic signatures. Here, we sought to identify signaling pathways using targeted RNA expression-based pathway analysis. Methods We analyzed 44 TNBC cases diagnosed at NYU Langone Health between March 2011 and April 2018. Samples were compared based on histology, immunohistochemistry, and epigenetics. We assessed mRNA expression levels of pathway-specific target genes with RT-qPCR using the OncoSIGNal pathway profiling assay. Signal transduction pathway analyses were used to measure activity of the estrogen receptor (ER), androgen receptor (AR), phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK), Hedgehog (HH), Notch, and transforming growth factor beta (TGF- β) pathways. Results DNA methylation Cluster 1, which is enriched for apocrine histology and PI3K/Akt mutations, showed no increased activity in PI3K pathway by RNA expression, but showed increase of AR and MAPK signaling, with a single case of increased ER signaling in the absence of ESR1 activating mutations. In contrast, both DNA methylation Cluster 2 (no recurrent mutations) and Cluster 3 (DNA instability cluster) showed upregulation in PI3K pathway in 4/7 (57%) and 11/25 (44%) of cases, respectively. PI3K pathway activation was invariably associated with high Ki-67 >15%, however not all high Ki-67 cases showed high PI3K activity. Invasive ductal carcinoma (IDC) morphology was characterized by a nearly even split between MAPK (13/28, 46%) and PI3K (12/28, 44%), with single HH and TGF- β driven cases. In 5/28 (18%) of IDC we did not identify an upregulated pathway. Discussion We show that RNA expression-based pathway activity and mutational and epigenetic analyses provide complimentary information about the molecular landscape of TNBC. PI3K mutations are early drivers and lead to distinct epigenetic signatures, however they are not associated with increased PI3K pathway activity by RNA expression. In contrast, TNBC without PI3K mutations may show activation of the PI3K pathway in the absence of PI3K/Akt mutations. Our observation suggests that distinct biomarkers may be differently suited to predict response to PI3K inhibitors in TNBC. This may include expanding the number of patients for which PI3K/Akt pathway inhibition might be a therapeutic option, as well as explaining the lack of response in PI3K mutated tumors. Citation Format: Chanel Schroff, Lawrence Lin, Dianne van Strijp, Daniel Roses, Sigi Neerken, Freya Schnabel, Farbod Darvishian, Matija Snuderl. Identifying mutation-independent signaling pathway activation in molecular subtypes of triple negative breast cancers [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 PO4-26-06.

Abstract ED10-02: Identifying Collateral Vulnerabilities in the AKT pathway for Therapeutic Benefit

Abstract The PI 3-kinase (PI3K) and AKT signaling pathway plays a critical role in regulating all aspects of normal cellular physiology, and is also frequently deregulated in human pathophysiologies, most evidently in cancer and diabetes. Three AKT isoforms exist in humans encoded by distinct genes (AKT1, AKT2, AKT3), and although originally thought to function redundantly, many studies have shown that AKT isoforms have non-overlapping and unique roles in both normal physiology and disease. Similarly, genetic lesions in the PIK3CA and AKT oncogenes have been described, and many of the genes that contribute to PI3K/AKT pathway activation and also signal termination have been found to be altered in human cancers. Numerous drugs that inhibit PI3K as well AKT have been developed for therapeutic use in patients, and many of these are being evaluated in late-stage clinical trials. During the seminar, I will review the major mechanisms that contribute to AKT hyperactivation in cancer. Genetic lesions in the PI3K/AKT pathway in human cancers will also be discussed, as well as efforts to target this pathway therapeutically, including our recent efforts aimed at developing new AKT degrader compounds. I will further present new studies using CRISPR-based genome wide synthetic lethal screens to identify genes that synergize with PI3K and AKT pathway inhibition to induce a cytotoxic response in TNBC addicted to pathway hyperactivation. I will present studies on genes and mechanisms that represent collateral vulnerabilities to pathway inhibition and our efforts at designing combination therapy strategies for cancer patients with genetic aberrations in the PI3K and AKT pathway. Citation Format: A. Toker. Identifying Collateral Vulnerabilities in the AKT pathway for Therapeutic Benefit [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 ED10-02.

Abstract PS17-06: A phase I trial of the PI3K inhibitor (PI3Ki) copanlisib and fulvestrant in combination with continuous or intermittent abemaciclib in patients with estrogen receptor-positive (ER+), HER2-negative (HER2-) metastatic breast cancer

Abstract Background: Activation of the phosphoinositide 3-kinase (PI3K) pathway is a prominent resistance mechanism to endocrine therapy and CDK4/6 inhibition (CDK4/6i) in ER+, HER2- breast cancer. There is reciprocal crosstalk between PI3K and cell cycle regulatory pathways. Triplet therapy that targets ER, CDK4/6, and PI3K prevents or delays disease progression in preclinical models of ER+, HER2- breast cancer. However, the toxicities of triplet therapy hinder its clinical development. Copanlisib (COP) is an intravenous (IV) PI3Ki with potent activity against α and δ isoforms. Intermittent high-dose PI3Ki is expected to be less toxic and more effective than continuous daily dosing. We set out to determine the recommended phase 2 dose (RP2D) for the COP/Fulvestrant (FUL)/Abemaciclib (ABE) combination. Methods: Eligible patients (pts) included men or women with ER+, HER2- metastatic breast cancer (MBC), with no more than 1 prior chemotherapy in the metastatic setting, and no limits on prior endocrine or targeted therapy. Prior FUL, CDK4/6i and PI3K/mTORi were allowed. Pts with HbA1c >8.5% were excluded. Pts received FUL 500 mg IM standard dosing and COP/ABE at assigned doses. Premenopausal women also received a GnRH agonist. Dose-limiting toxicity (DLT) was assessed during the first 28-day cycle. Adverse events (AEs) were graded according to NCI-CTCAE v5.0. Dose escalation/de-escalation was based on continual reassessment (CRM) and the RP2D was defined as the dose with the highest probability of the DLT rate within the acceptable 25~35%. Results: Between June 2020 and June 2023, 24 pts with ER+, HER2- MBC were enrolled. Table 1 shows the dose levels (DLs) and DLTs. The first 10 pts were enrolled to Part A on two consecutive DLs of COP (DL1: 45 mg IV D1 and D15, n=7; DL2: 45 mg IV D1, D8, and D15, n=3), with ABE fixed at 100 mg PO BID continuously. DL2a exceeded the maximum tolerated dose (MTD). DL1a was tolerable based on Cycle 1 AEs. However, during subsequent cycles, only 1 of the 7 pts in DL1a maintained the intended dose of ABE due to neutropenia, others reduced ABE to 50mg bid (n=5) or discontinued (n=1). This triggered a protocol amendment, adding Part B with ABE administered 5 days on and 2 days off (5-on/2-off) intermittent schedule. 14 pts were enrolled to Part B in 2 consecutive DLs of COP (DL1b: n=7; DL2b: n=7), the same as in Part A. ABE was fixed at 100 mg PO Bid 5-on/2-off. DL2b exceeded the MTD. DL1b was tolerable based on Cycle 1 AEs. In addition, in subsequent cycles, only 1 of 7 pts on DL1b required dose reduction of ABE to 50mg bid. RP2D is therefore defined as DL1b. Grade (G) 4 AEs were rare. The most common all cycle G3 AEs included transient hypertension (45%), rash (30%), anemia (25%), neutropenia (25%), and AST elevation (20%). As of 6/14/2023, 22 pts, with a median of 1 prior metastatic regimen (range 0-5), prior CDK4/6i in 18 (82%), liver mets in 8 (36%), and PIK3CA mutation in 8 (50%) of 16 pts with known mutation status, were evaluable for response. There were 5 partial responses and 3 stable diseases lasting for ≥24 weeks. Clinical benefit rate was 36.4% (8/22, 95%CI: 17.19% ~ 59.34%). Responses were observed in pts regardless of prior CDK4/6i, FUL, or PIK3CA mutation. Conclusion: COP/FUL/intermittent ABE can be safely administered with fair overall tolerability. Preliminary anti-tumor activity was observed, which will be further examined in the randomized phase II trial (NCT 03939897). Table 1 Dose-limiting Toxicities by Dose Level Citation Format: Cynthia Ma, Jingqin Luo, Jessica Moss, Maryann Kwa, Ritesh Parajuli, Katia Khoury, Emily Douglas, Ashley Frith, Caron Rigden, Foluso Ademuyiwa, Shana Thomas, Brittney Haas, Gerburg Wulf, Claire Dees, Rabih Said. A phase I trial of the PI3K inhibitor (PI3Ki) copanlisib and fulvestrant in combination with continuous or intermittent abemaciclib in patients with estrogen receptor-positive (ER+), HER2-negative (HER2-) metastatic 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 PS17-06.

Abstract PO3-02-03: Ketogenic diet and letrozole to inhibit PI3K signaling in ER+ breast cancer

Abstract Obesity is associated with an increased risk of breast cancer recurrence, similar in magnitude to the reduction in risk seen with the use of adjuvant chemotherapy. However, whether and how obesity causes that increased risk of recurrence remains unknown. Plausible mechanisms include obesity-mediated increases in estrogen signaling, inflammatory conditions resulting from obesity, or effects on insulin or other growth factor signaling pathways resulting from the insulin-resistant state that often accompanies obesity. While phosphatidyl inositol 3-kinase (PI3K) signaling is the normal, biological effector of insulin signaling, abnormal activation of PI3K is of particular concern in breast cancer, where it is associated with resistance to endocrine therapies and HER2-targeted therapies. We hypothesize that at least part of the adverse outcome associated with obesity results from aberrant activation of PI3K signaling in tumors. This may confer resistance to established therapies, or directly stimulate tumor growth (particularly in tumors with existing PI3K pathway mutations), or both. We tested a dietary intervention designed to alter insulin resistance pathophysiology coupled with endocrine therapy in the well-established neoadjuvant treatment paradigm in breast cancer. This will allow us to study the effects of the dietary intervention directly on tumor biology. The primary objective of this neoadjuvant study was to assess feasibility and tolerability of 2 weeks of a very low-carbohydrate ketogenic diet in combination with letrozole for patients with early stage operable ER+ disease. Up to 36 patients will be enrolled for this pilot and feasibility study (24 in the treatment group and 12 controls). Baseline metabolic parameters will be measured and the treatment group will begin a dietitian-supervised 2-week diet to induce a ketogenic state, along with letrozole 2.5 mg daily. The control group will receive only letrozole. At the end of 2 weeks, patients will proceed with surgical treatment of their breast cancer. We will report the primary outcome measure of feasibility, assessed by determining 1) the proportion of patients in the diet group achieving ketosis, 2) adherence to the diet and 3) participant-reported measures of stress, fatigue, and emotional health. For both groups, cell proliferation will be measured in a tumor biopsy obtained from the surgical specimen and compared with the pre-treatment diagnostic biopsy. Correlative studies will evaluate tumor markers of insulin/PI3K signaling before and after the intervention and between diet and control. Citation Format: Brent Rexer, Vandana Abramson, Sara Nunnery, Sonya Reid, Laura Kennedy, Melinda Sanders, Jessica Bennett, Paula Gonzalez-Ericsson, Violeta Sanchez. Ketogenic diet and letrozole to inhibit PI3K signaling in ER+ 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 PO3-02-03.

Molecular targets of glucocorticoids that elucidate their therapeutic efficacy in aggressive lymphomas

Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK.

Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer

Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.

PI3Kγδ inhibition suppresses key disease features in a rat model of asthma

BackgroundTwo isoforms of Phosphoinositide 3-kinase (PI3K), p110γ and p110δ, are predominantly expressed in leukocytes and represent attractive therapeutic targets for the treatment of allergic asthma. The study aim was to assess the impact of administration of an inhaled PI3Kγδ inhibitor (AZD8154) in a rat model of asthma.MethodsFirstly, we checked that the tool compound, AZD8154, inhibited rat PI3K γ & δ kinases using rat cell-based assays. Subsequently, a time-course study was conducted in a rat model of asthma to assess PI3K activity in the lung and how it is temporally associated with other key transcription pathways and asthma like features of the model. Finally, the impact on lung dosed AZD8154 on target engagement, pathway specificity, airway inflammation and lung function changes was assessed.ResultsData showed that AZD8154 could inhibit rat PI3K γ & δ isoforms and, in a rat model of allergic asthma the PI3K pathway was activated in the lung. Intratracheal administration of AZD8154 caused a dose related suppression PI3K pathway activation (reduction in pAkt) and unlike after budesonide treatment, STAT and NF-κB pathways were not affected by AZD8154. The suppression of the PI3K pathway led to a marked inhibition of airway inflammation and reduction in changes in lung function.ConclusionThese data show that a dual PI3Kγδ inhibitor suppress key features of disease in a rat model of asthma to a similar degree as budesonide and indicate that dual PI3Kγδ inhibition may be an effective treatment for people suffering from allergic asthma.

The PI3K/AKT/mTOR signaling pathway in breast cancer: Review of clinical trials and latest advances.

Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer mortality in women. As the phosphatidylinositol 3-kinase (PI3K) signaling pathway is involved in a wide range of physiological functions of cells including growth, proliferation, motility, and angiogenesis, any alteration in this axis could induce oncogenic features; therefore, numerous preclinical and clinical studies assessed agents able to inhibit the components of this pathway in BC patients. To the best of our knowledge, this is the first study that analyzed all the registered clinical trials investigating safety and efficacy of the PI3K/AKT/mTOR axis inhibitors in BC. Of note, we found that the trends of PI3K inhibitors in recent years were superior as compared with the inhibitors of either AKT or mTOR. However, most of the trials entering phase III and IV used mTOR inhibitors (majorly Everolimus) followed by PI3K inhibitors (majorly Alpelisib) leading to the FDA approval of these drugs in the BC context. Despite favorable efficacies, our analysis shows that the majority of trials are utilizing PI3K pathway inhibitors in combination with hormone therapy and chemotherapy; implying monotherapy cannot yield huge clinical benefits, at least partly, due to the activation of compensatory mechanisms. To emphasize the beneficial effects of these inhibitors in combined-modal strategies, we also reviewed recent studies which investigated the conjugation of nanocarriers with PI3K inhibitors to reduce harmful toxicities, increase the local concentration, and improve their efficacies in the context of BC therapy.

Abstract 5841: A genome wide CRISPR-Cas9 screen identifies mediators of resistance to dual PI3K/mTOR inhibition in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor with a devastating 5-year survival rate of 6.8%. The current standard of care, consisting of surgery, radiation, and temozolomide (TMZ), has remained unchanged for over the past 15 years despite its limited efficacy and the serious therapy-related adverse events associated to this regimen. Although some frequent genetic alterations have been identified in GBM, no targeted therapies have shown efficacy in these patients to date. Among these, the phosphoinositide 3-kinase (PI3K) pathway is a signaling network that regulates cell growth, proliferation, migration, and invasion. Extensive molecular characterization of GBM has identified alterations in the components of the PI3K pathway in 80% of primary GBMs, including amplification of receptor tyrosine kinases (RTKs), activating mutations in the PI3K catalytic subunit (PIK3CA), loss-of-function mutations in the PI3K regulatory subunit (PIK3R1), and loss of the major tumor suppressor of the pathway, phosphatase and tensin homologue (PTEN). Despite the universal upregulation of PI3K pathway signaling associated with these genetic alterations, clinical trials investigating PI3K inhibitors in GBM have failed due to insufficient antitumor activity and dose-limiting toxicities. To elucidate mechanisms of resistance to PI3K inhibition in GBM, we used a genome-wide functional CRISPR-Cas9 knockout screen to identify genes that mediate resistance to the dual PI3K/mTOR inhibitor XL765 in PTEN-null SF295 GBM cells. We identified reactivation of the PI3K pathway and inhibition of autophagy as potential resistance mechanisms to dual PI3K/mTOR inhibition. Using orthogonal approaches, we will functionally validate our candidate hits through mechanistic studies, with patient-derived models, and with in vivo orthotopic xenografts. Our findings will elucidate novel mechanisms of resistance to PI3K/mTOR inhibition in GBM and will streamline rational drug combinations aimed to overcome resistance, thus eventually maximizing therapeutic responses to PI3K inhibitors in GBM. Citation Format: Annalisa V. Ferrotta, Arianna Izawa-Ishiguro, Meaghan Grogan, Ingo K. Mellinghoff. A genome wide CRISPR-Cas9 screen identifies mediators of resistance to dual PI3K/mTOR inhibition in glioblastoma multiforme [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 5841.

Abstract 4131: Combination therapeutic strategy to fight against triple-negative breast cancer

Abstract Triple-negative breast cancers (TNBCs) lack estrogen-and progesteron receptors and human epidermal growth factor receptor-2 (HER2) which limits the therapeutic approaches. The high metastasizing capability of TNBCs is in correlation with the poor oxygenation of the cancerous breast tissue. This leads to the activation of the hypoxia-inducible-factor 1 (HIF-1), which induces multiple target genes that are involved in tumor progression and metastasis formation. This makes HIF-1 an attractive drug target, however, HIF-1 inhibitors failed if applied in monotherapy. Therefore, our aim is to investigate the combined therapeutic effect of HIF-1 inhibitor acriflavine and paclitaxel, a commonly used taxane in TNBC treatment. The synergy assessment of the two drugs was carried out by utilizing Sulforhodamine B assay on TNBC cell lines. The in vivo toxicity and efficacy of the drug combination was tested on NOD SCID mice with orthotopically inoculated MDA-MB-231 cell line. Quantitative and qualitative analysis of multiple genes involved in tumor progression was performed by mRNA and protein expression level assessment with qPCR, western blot and immunohistochemistry methods. HIF-1 stimulation was achieved by either incubating the cells at 1% oxygen level or through CoCl2 treatment. The in vitro mRNA expression studies demonstrated that acriflavin possesses a hypoxic-effect-mitigating property on the expression of metastasis-involved- and HIF-1 target genes. Acriflavine alleviated the epithelial-mesenchymal transition of TNBC cells by increasing the expression of the epithelial marker, E-cadherin. At protein level, we detected decrease in HIF-1alpha and Phospho-Protein kinase B (Akt) and increase in p53 tumor suppressor gene expression in response to acriflavine treatment. We further established MDA-MB-231 xenograft model in mice for investigating in vivo toxicity and efficacy. The drug combination efficiently reduced the tumor size and the macrometastasis formation compared to the control groups. The protein analysis of frozen tumor tissue samples indicated that the combination therapy decreased the HIF-1alpha expression and the amount of Phospho-Akt and Phospho-Mitogen-activated protein kinases (pMAPK/pERK) in CoCl2treated mice. Additionally, expression of epithelial markers increased and that of mesenchymal markers decreased. The combination therapy led to a decrease in the platelet-derived growth factor receptor beta (PDGFRB) as well. To conclude, we identified a novel drug combination which can efficiently combat TNBC by inhibiting HIF-1 signaling, mitigating cell migration and metastasis formation and suppressing the activation of the Phosphoinositide 3-kinase (PI3K) and MAPK pathways. This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Citation Format: Laura Svajda, Ivan Ranđelović, Sára Eszter Surguta, Marcell Baranyi, István Kenessey, Mihály Cserepes, József Tóvári. Combination therapeutic strategy to fight against triple-negative breast 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 4131.

Abstract 6448: Prevalence and spectrum of PIK3CA mutations in Indian breast cancer patients

Abstract Introduction: Breast carcinomas are the most commonly encountered cancers in women globally. Due to advancements in cancer research the death rate in breast cancer has reduced by 41% since 1989; however, the downward trend has recently slowed emphasizing on new breast carcinomas treatment discovery. Breast carcinomas are molecularly categorized by the expression of specific hormone receptors, as well as the overexpression of human epidermal growth factor receptor 2 (HER2). Approximately 80% of breast cancers have been termed HER2-negative, but researchers now recognize that 55% of all breast cancers may actually contain low levels of HER2, levels that are still targetable by HER2-directed therapies such as Trastuzumab Deruxtecan. The phosphoinositide 3-kinase (PI3K) pathway is the most frequently altered pathway in HR+/HER2- breast carcinomas and is associated with tumor development, disease progression, and endocrine resistance. Methodology: The study entailed histological verification of breast cancer, and thence the extraction of DNA from formalin fixed, paraffin embedded (FFPE) tissue samples. A tumor content of 20% was the cut-off for performing the PIK3CA mutation analysis. The DNA was subjected to mutation analysis using a FDA-approved PIK3CA RGQ Real Time PCR based assay that has ability to detect 11 mutations in the PIK3CA gene encompasses exons 7, 9, and 20. Results: A total of 696 breast cancer patient samples (with more than 20% neoplastic tissue content) were screened for PIK3CA mutations. Of these, 275 (39.5%) (Male: Female ratio = 1:26.5; Age range = 30-80 years) harbored a mutation of the PIK3CA gene. Females were commonly affected; however, the males were also found to have PIK3CA mutation. The most common affected age group is from 41 to50 years. The positivity rate exon wise were as follows: Exon 7 (1.45%), Exon 9 (39.27%), and Exon 20 (44.27%). There was presence of co-mutations amongst different exons, which comprised of 4%. HR+/HER2- (50%) followed by HR-/Her2- (3.27%) and HR+/HER2+ (1.82%). The HER2- cases (Score 1 and Score 2) by IHC were considered as low positive cases (14%). Conclusion: This is one of the largest study to report frequency of PIK3CA mutations in the Indian population. PIK3CA mutations are commonly encountered in hormone receptor positive and HER2 Negative metastatic breast cancer and have shown promising clinical outcome leading to better prognosis. It provides insights into genotyping each breast cancer and exploits the usage of PI3K/AKT inhibitors together with other combination of drugs for effective target treatment strategies. Keywords: Breast carcinomas, PIK3CA, HR/HER2, Targeted Therapeutic Citation Format: Rahul Katara, Tina Bhardwaj, Monika Rawat, Deepak K. Sharma, Vipin Kumar, Shivani Sharma, Vaishali Dagar, Pawan Kumar, Sambit K. Mohanty. Prevalence and spectrum of PIK3CA mutations in Indian breast cancer patients [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 6448.

The human adenovirus PI3K-Akt activator E4orf1 is targeted by the tumor suppressor p53.

Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.IMPORTANCEHuman adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.

Abstract B028: Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia

Abstract Somatic mutations in the phosphatidylinositol 3-kinase (PI3K) pathway occur in 84% of endometrial carcinomas (EC) and up to 68% of endometrial hyperplasia (EH). Regulation of PI3K/Akt/mTOR signaling is integral for normal endometrial function and is governed by tumor suppressor genes PTEN and PIK3R1 and proto-oncogene PIK3CA. While the actions of PTEN and PIK3CA are in direct opposition as a phosphatase and kinase, respectively, PIK3R1 regulates PI3K/Akt/mTOR signaling in two distinct ways. As the regulatory subunit of the PI3K enzyme, PIK3R1 controls the catalytic activity of PIK3CA and augments PTEN activity. Mutations or alterations in PIK3R1 are present in 33% of EC and 9% of EH, the majority of which lead to loss of PIK3R1 protein (p85a). Both loss of PTEN and PIK3R1 lead to increased proliferation through PI3K/Akt/mTOR signaling, but the structural, functional, and mechanistic implications of PIK3R1 loss in the endometrial epithelium (EE) in vivo are largely unexplored. To further interrogate the changes in the endometrium resulting from PIK3R1 loss, we have created a murine model of endometrial epithelial-specific PIK3R1 loss driven by Pax8-Cre. Mice were estrus cycle staged and sacrificed in the diestrus phase to reduce confounding variables when assessing phenotypes between genotypes. PIK3R1 loss was confirmed by immunohistochemistry (IHC). Histology and immunostaining of control and PIK3R1 knockout (KO) mice were assessed by IHC and immunofluorescence. We found that PIK3R1 loss in the EE leads to EH distinct from what is observed with PTEN loss. In contrast to the EH with atypia seen In PTEN KO mice, the EH resulting from PIKR1 loss more closely resembles EH without atypia. The endometrium of PIK3R1 KO mice is characterized by thickened luminal epithelia with jagged edges and irregularities, mild glandular crowding, and occasional nuclear atypia. PIK3R1 KO mice also showed an increase in proliferation (Ki-67) and downstream PI3K signaling (phospho-S6) in the EE. Gene expression changes occurring within the EE from PIK3R1 KO and Cre-negative control mice were also assessed by RNA-seq. In vivo genomic analyses were compared to in vitro knockdown of PIK3R1 and publicly available patient data. We found that PIK3R1 loss leads to an increase in signaling through the PI3K/Akt/mTOR and MAPK pathways. Our data show that the effect of PIK3R1 loss is sufficient to cause EH and may have shared and distinct actions from PTEN. Citation Format: Shannon K. Harkins, Hilary J. Skalski, Amelia Arendt, Laura Pavliscak, Mike R. Wilson, Ronald L. Chandler. Epithelial-specific loss of PIK3R1 in a murine model result in endometrial hyperplasia [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B028.

Renal Metabolomics Study and Critical Pathway Validation of Shenkang Injection in the Treatment of Chronic Renal Failure.

To reveal the mechanism of Shenkang injection (SKI) in the treatment of chronic renal failure, and verify the key pathway. In this work, an untargeted metabolomics approach was performed by LC-MS coupled with multivariate statistical analysis to provide new insights into therapeutic mechanism of SKI. Hematoxylin-eosin (H&E) Staining and Immunohistochemistry were used to evaluate the effects of drug treatment, Western blot was used to verify the critical pathway. Then, a total of 44 potential biomarkers of chronic renal failure (CRF) were identified and reversed regulation, including 2,8-dihydroxypurine, 5-methoxytryptophan, uric acid, acetylcarnitine, taurine, etc. Mainly concerned with arginine and proline metabolism, purine metabolism, histidine metabolism, etc. Pathological examination showed that the renal interstitium of SKI group was significantly improved, with fewer inflammatory cells and thinner vascular walls compared with the model group. Immunohistochemical results showed that the expression of α-smooth muscle actin (α-SMA) was decreased, and the expression of E-cadherin was increased in CRF model group, and the two indicators were reversed regulation in SKI injection, indicating that the degree of fibrosis was relieved. Critical signaling pathway phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and nuclear factor-kappaB (NF-κB) protein expressions were significantly inhibited. This study was the first to employ metabolomics to elucidate the underlying mechanisms of SKI in chronic renal failure. The results would provide some support for clinical application of traditional Chinese medicines in clinic.

NIPBL-mediated RAD21 facilitates tumorigenicity by the PI3K pathway in non-small-cell lung cancer

It is urgent to identify novel early diagnostic markers and therapeutic targets for non-small-cell lung cancer (NSCLC), which accounts for 85% of lung cancer cases and has a 5-year survival rate of 4–17%. Here, chromatin immunoprecipitation (ChIP) was used to identify DNA‒protein interactions, RNA methylation was determined by methylated RNA immunoprecipitation (MeRIP), RNA stability was tested by an RNA decay assay. We showed that RAD21, a member of the cohesin complex, is upregulated in NSCLC tissues and cell lines and found to be an independent prognostic factor for overall survival (OS) of NSCLC patients. Mechanistically, the cohesin loading factor Nipped-B-Like Protein (NIPBL) promoted RAD21 gene transcription by enhancing histone H3 lysine 27 (H3K27) demethylation via recruiting lysine demethylase 6B (KDM6B) to the RAD21 gene promoter. RAD21 enhanced phosphatidylinositol 3-kinase (PI3K) gene transcription, and NIPBL reversed the effect of enhancer of zeste 2; catalytic subunit of polycomb repressive complex 2 (EZH2) on RAD21-mediated PI3K gene transcription by disrupting the association between EZH2 and RAD21. Moreover, NIPBL level was increased by stabilization of its transcripts through mRNA methylation. These findings highlight the oncogenic role of RAD21 in NSCLC and suggest its use as a potential diagnostic marker and therapeutic target for NSCLC.

Simultaneous inhibition of PI3K and PAK in preclinical models of neurofibromatosis type 2-related schwannomatosis

Neurofibromatosis Type 2 (NF2)-related schwannomatosis is a genetic disorder that causes development of multiple types of nervous system tumors. The primary and diagnostic tumor type is bilateral vestibular schwannoma. There is no cure or drug therapy for NF2. Recommended treatments include surgical resection and radiation, both of which can leave patients with severe neurological deficits or increase the risk of future malignant tumors. Results of our previous pilot high-throughput drug screen identified phosphoinositide 3-kinase (PI3K) inhibitors as strong candidates based on loss of viability of mouse merlin-deficient Schwann cells (MD-SCs). Here we used novel human schwannoma model cells to conduct combination drug screens. We identified a class I PI3K inhibitor, pictilisib and p21 activated kinase (PAK) inhibitor, PF-3758309 as the top combination due to high synergy in cell viability assays. Both single and combination therapies significantly reduced growth of mouse MD-SCs in an orthotopic allograft mouse model. The inhibitor combination promoted cell cycle arrest and apoptosis in mouse merlin-deficient Schwann (MD-SCs) cells and cell cycle arrest in human MD-SCs. This study identifies the PI3K and PAK pathways as potential targets for combination drug treatment of NF2-related schwannomatosis.

Abstract A052: Dissecting the immune regulatory mechanisms of distinct PI3K alterations in invasive lobular carcinoma

Abstract Immunotherapy has been far less effective in breast cancer compared to other cancer types, with only a small fraction of patients having clinical benefit. Historically, invasive lobular carcinomas (ILC), the second most common histological subtype of breast cancer, have been categorized as immunological cold tumors, but recent studies suggest that ILCs may recruit a myeloid-rich immunosuppressive immune landscape. Cancer cell intrinsic features such as the genetic makeup of tumor cells can dictate the spatial distribution, composition and activation state of immune cells, which can exert both anti-tumor and pro-tumor activities and affect disease progression and immunotherapy response. Genetic aberration in key genes of the phosphoinositide-3 kinase (PI3K) signaling pathway are observed in >50% of primary ILCs. The overall aim of this project is to determine whether genetic aberrations in key genes of the PI3K pathway regulate the composition and functional state of the immune landscape, and whether this influences the clinical benefit of immunomodulatory strategies in ILC patients. To dissect the immune regulatory mechanisms of distinct PI3K alterations we have used transgenic ILC mouse models, in which immune profiling studies by flow cytometry and immunohistochemistry revealed that distinct PI3K alterations in cancer cells differentially affect the tumor immune landscape. With some mutations driving a myeloid-rich tumor microenvironment, whereas the immune landscape of tumors harboring other PI3K mutations is dominated by lymphoid cells. To understand the observed cancer genotype-immunophenotypes mechanistically, we have performed single-cell RNA sequencing and secretome analysis to assess cellular communication between the cancer cells and the immune system. We identified key differences in ligand-receptor interactions and secreted signaling that can explain the influx of different immunosuppressive immune cell types in the tumor microenvironment of murine ILCs. We are currently investigating whether therapeutic targeting of immunosuppression is a feasible strategy for PI3K-altered ILC tumors with a tumor-promoting immune landscape. Shedding light on the mechanisms that govern the diversity and function of immune cells in the tumor microenvironment will provide a greater understanding of interpatient heterogeneity, and will pave the way for the development of novel and more tailored immunomodulatory strategies for ILC patients. Citation Format: Antoinette van Weverwijk, Michael Schubert, Joseph Siefert, Martine van Miltenburg, Kim Vrijland, Julia Houthuijzen, Anne Stunnenberg, Cheei-Sing Hau, Lodewyk Wessels, Jos Jonkers, Karin E de Visser. Dissecting the immune regulatory mechanisms of distinct PI3K alterations in invasive lobular carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A052.

Effect of Sonic hedgehog gene-modified bone marrow mesenchymal stem cells on graft-induced retinal gliosis and retinal ganglion cells survival in diabetic mice.

To investigate the effects of Sonic hedgehog (Shh) gene-modified bone marrow mesenchymal stem cells (MSCs) on graft-induced retinal gliosis and retinal ganglion cells (RGCs) survival in diabetic mice. Bone marrow-derived MSCs were genetically modified with the Shh gene to generate a stably transfected cell line of Shh-modified MSCs (MSC-Shh). Intravitreal injections of MSC-Shh and green fluorescent protein-modified MSCs (MSC-Gfp; control) were administered in diabetic mice. After 4wk, the effects of MSC-Shh on retinal gliosis were evaluated using fundus photography, and markers of gliosis were examined by immunofluorescence and Western blotting. The neurotrophic factors expression and RGCs survival in the host retina were evaluated using Western blotting and immunofluorescence. The mechanisms underlying the effects of MSC-Shh was investigated. A significant reduction of proliferative vitreoretinopathy (PVR) was observed after intravitreal injection of MSC-Shh compared to MSC-Gfp. Significant downregulation of glial fibrillary acidic protein (GFAP) was demonstrated in the host retina after MSC-Shh administration compared to MSC-Gfp. The extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (AKT) and phosphatidylin-ositol-3-kinase (PI3K) pathways were significantly downregulated after MSC-Shh administration compared to MSC-Gfp. Brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) levels were significantly increased in the host retina, and RGCs loss was significantly prevented after MSC-Shh administration. MSC-Shh administration reduces graft-induced reactive gliosis following intravitreal injection in diabetic mice. The ERK1/2, AKT and PI3K pathways are involved in this process. MSC-Shh also increases the levels of neurotrophic factors in the host retina and promoted RGCs survival in diabetic mice.