Activation Of PI3K Pathway Research Articles

336-OR: Mandarin Fish Ranavirus Viral Insulin/IGF-Like Peptide Inhibits Human IGF-1 Receptor

Our recent studies published in PNAS and Molecular Metabolism showed for the very first time that viruses possess genes encoding viral insulin/IGF-1 like peptides (VILPs). These two studies provided several important insights on VILPs: 1. Identified VILPs in six different Iridoviridae viruses 2. Showed that chemically synthesized single chain (sc, IGF-1 like) and double chain (dc, insulin-like) VILPs can bind to human insulin and IGF-1 receptors and stimulate downstream signaling. 3. Demonstrated that VILPs can increase glucose uptake in vitro and in vivo. 4. Identified the unique tissue-specific effects of VILPs. 5. Found sequences of these viruses in the human gut and plasma virome. In the present study, we focused on a novel, Mandarin Fish Ranavirus (MFRV) VILP and characterized both sc and dc forms for the first time. MFRV scVILP binds to human IGF1R with an affinity 100x higher than insulin, making it a VILP with the highest affinity to this receptor so far discovered. MFRV scVILP also activates IGF1R phosphorylation and post-receptor signaling. Interestingly, although MFRV scVILP is a weak agonist of IGF1R, it inhibits IGF-1 signaling at higher concentrations. When we treat cells with both IGF-1 and MFRV VILP, MFRV scVILP inhibits IGF-1 (10nM) stimulated IGF1R phosphorylation and activation of PI3K and MAPK pathways at high concentrations (> 250 nM). Surprisingly, the dc version of MFRV VILP neither bind nor stimulate IGF1R. This suggests that the C-chain of MFRV VILP is essential for both binding to IGF1R and the IGF-1 inhibitory effects. Taken together, our results revealed that MFRV scVILP is a novel antagonist/inhibitor of IGF-1R. IGF1R signaling is overly activated in breast, prostate and colorectal cancers. Despite tremendous efforts to develop an effective inhibitor of IGF1R, these attempts failed. MFRV VILP has the potential to contribute development of new therapeutics in cancer therapy and to help us better understand ligand-receptor interactions and mechanisms of IGF1R inhibition. Disclosure M. Chrudinova: None. V. Gelfanov: Employee; Self; Novo Nordisk, Employee; Spouse/Partner; Eli Lilly and Company. L. Zakova: None. J. Cutone: None. A. Powis: None. K. Sevgi: None. J. Jiracek: None. R. Dimarchi: Employee; Self; Novo Nordisk. E. Altindis: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (K01DK117967)

Identification of signal transduction pathway activity with potential clinical target in high-grade serous ovarian and tubal carcinoma.

e17541 Background: High-grade serous carcinoma (HGSC) is the most common subtype of epithelial ovarian and tubal cancer. It has a high mortality rate, even after successful first-line treatment with debulking surgery and chemotherapy. Although therapeutic options for targeted therapy are rapidly expanding, identification of patients who respond to these therapies remains a challenge. In recognition of the importance of the functional phenotype of cancer cells, Verhaegh et al. (Cancer Res 2014) developed assays to measure functional activity of signal transduction pathways (STPs) based on mRNA expression levels of pathway-specific target genes. In this study, we aimed to identify HGSCs with STP activity with potential clinical target by comparing their activity assessment with STP activity in normal Fallopian tube epithelium (FTE), the tissue of origin of most HGSCs. Methods: We included 67 primary tumor samples taken prior to start of chemotherapy of postmenopausal patients diagnosed with advanced stage HGSC and 8 morphologically normal FTE samples of healthy postmenopausal women. Using OncoSignal pathway assays, we assessed functional pathway activity of the androgen receptor (AR), estrogen receptor (ER), PI3K, Hedgehog, TGF-𝛽 and Wnt pathways. Differences in STP activity between groups were compared with Mann-Whitney U tests. Cut-off value for aberrant STP activity was defined as two standard deviations above the mean value of STP activity measured in FTE samples. Results: In the HGSC group we observed lower median ER (p < 0.001) and Wnt (p = 0.046) pathway activity and higher median PI3K (p = 0.025) and TGF-𝛽 pathway (p = 0.030) activity as compared to the normal FTE group. In individual HGSC samples, aberrant activity was identified for the AR (n = 14), PI3K (n = 16), Hedgehog (n = 4), TGF-𝛽 (n = 36) and Wnt (n = 10) pathways. Frequently observed combinations of aberrant STP activity were AR/TGF-𝛽 (n = 8), TGF-𝛽/Wnt (n = 6) and PI3K/Hedgehog (n = 3). In total, we identified at least one STP with potential clinical target in 52 of the 67 HGSC samples. Conclusions: Our analysis enabled the identification of STP activity with potential clinical target in 78% of the analyzed HGSC samples. Differentiation between normal and aberrant STP activity could have clinical utility in the selection of HGSC patients for targeted therapy.

Rotundic acid reduces LPS-induced acute lung injury in vitro and in vivo through regulating TLR4 dimer.

Acute lung injury (ALI) is a serious clinical disease. Rotundic acid (RA), a natural ingredient isolated from Ilex rotunda Thunb, exhibits multiple pharmacological activities. However, RA's therapeutic effect and mechanism on ALI remain to be elucidated. The present study aimed to further clarify its regulating effects on inflammation in vitro and in vivo. Our results indicated that RA significantly inhibited the overproduction of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). RA decreased ROS production and calcium influx. In addition, RA inhibited the activation of PI3K, MAPK, and NF-κB pathways and enhanced the activity of nuclear factor E2-related factor 2 (Nrf2) signaling. The cellular thermal shift assay and docking results indicated that RA bind to TLR4 to block TLR4 dimerization. Furthermore, RA pretreatment effectively inhibited ear edema induced by xylene and LPS-induced endotoxin death and had a protective effect on LPS-induced ALI. Our findings collectively indicated that RA has anti-inflammatory effects, which may serve as a potential therapeutic option for pulmonary inflammation.

PTEN loss correlates with T cell exclusion across human cancers

BackgroundRecent evidences had shown that loss in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was associated with immunotherapy resistance, which may be attributed to the non-T-cell-inflamed tumor microenvironment. The impact of PTEN loss on tumor microenvironment, especially regarding T cell infiltration across tumor types is not well understood.MethodsUtilizing The Cancer Genome Atlas (TCGA) and publicly available dataset of immunotherapy, we explored the correlation of PTEN expressing level or genomic loss with tumor immune microenvironment and response to immunotherapy. We further investigated the involvement of PI3K-AKT-mTOR pathway activation, which is known to be the subsequent effect of PTEN loss, in the immune microenvironment modulation.ResultsWe reveal that PTEN mRNA expression is significantly positively correlated with CD4/CD8A gene expression and T cells infiltration especially T helpers cells, central memory T cell and effector memory T cells in multiples tumor types. Genomic loss of PTEN is associated with reduced CD8+ T cells, type 1 T helper cells, and increased type 2 T helper cells, immunosuppressed genes (e.g. VEGFA) expression. Furthermore, T cell exclusive phenotype is also observed in tumor with PI3K pathway activation or genomic gain in PIK3CA or PIK3CB. PTEN loss and PI3K pathway activation correlate with immunosuppressive microenvironment, especially in terms of T cell exclusion. PTEN loss predict poor therapeutic response and worse survival outcome in patients receiving immunotherapy.ConclusionThese data brings insight into the role of PTEN loss in T cell exclusion and immunotherapy resistance, and inspires further research on immune modulating strategy to augment immunotherapy.

The metastasis suppressor protein NM23-H1 modulates the PI3K-AKT axis through interaction with the p110\u03b1 catalytic subunit

The PI3K pathway is one of the most deregulated pathways in cancer, which is predominantly due to gain of function mutations or altered expression of the PI3KCA gene. This is codified by what is seen for the class I PI3K catalytic subunit p110α, a common feature of many cancers. The metastasis suppressor protein NM23-H1 (NME1), whose ability to suppress the metastasis activities of different tumors has been widely described and was previously reported to alter phosphatidylinositol signaling. Here, we show interaction of NM23-H1 with the p110α subunit and the functional consequence of this interaction. This interaction is predominantly localized at the plasma membrane with some signals seen in the cytoplasmic compartment. Analysis of NM23-H1 levels showed a negative correlation between NM23-H1 expression and Akt phosphorylation, the key marker of PI3K pathway activation. Investigating the functional consequence of this interaction using cell motility and clonogenicity assays showed that expression of NM23-H1 reversed the enhanced migration, invasion, adhesion, and filopodia structure formation in cells expressing the p110α catalytic subunit. A similar trend was seen in anchorage-independent assays. Notably, differential analyses using NM23-H1 mutants which lacked the enzymatic and metastasis suppressor activity, showed no detectable interaction between p110α and the NM23-H1 mutant proteins P96S, H118F, and S120G, as well as no dysregulation of the PI3K-AKT axis.

Proteomic Resistance Biomarkers for PI3K Inhibitor in Triple Negative Breast Cancer Patient-Derived Xenograft Models.

Simple SummaryThe objective of this study is to identify potential proteomic biomarkers in triple negative breast cancer (TNBC) that associate with response to PI3K inhibitors which are in clinical trials. We tested a panel of TNBC patient-derived xenograft (PDX) models for their tumor growth response to a pan-PI3K inhibitor, BKM120. Proteomic analyses by reverse phase protein array (RPPA) of 182 markers were performed on baseline and post short-term treatment PDX samples, to correlate with tumor growth response. We identified several baseline and treatment induced proteomic biomarkers in association with resistance. These results provide important insights for the development of PI3K inhibitors in TNBC. PI3K pathway activation is frequently observed in triple negative breast cancer (TNBC). However, single agent PI3K inhibitors have shown limited anti-tumor activity. To investigate biomarkers of response and resistance mechanisms, we tested 17 TNBC patient-derived xenograft (PDX) models representing diverse genomic backgrounds and varying degrees of PI3K pathway signaling activities for their tumor growth response to the pan-PI3K inhibitor, BKM120. Baseline and post-treatment PDX tumors were subjected to reverse phase protein array (RPPA) to identify protein markers associated with tumor growth response. While BKM120 consistently reduced PI3K pathway activity, as demonstrated by reduced levels of phosphorylated AKT, percentage tumor growth inhibition (%TGI) ranged from 35% in the least sensitive to 84% in the most sensitive model. Several biomarkers showed significant association with resistance, including elevated baseline levels of growth factor receptors (EGFR, pHER3 Y1197), PI3Kp85 regulatory subunit, anti-apoptotic protein BclXL, EMT (Vimentin, MMP9, IntegrinaV), NFKB pathway (IkappaB, RANKL), and intracellular signaling molecules including Caveolin, CBP, and KLF4, as well as treatment-induced increases in the levels of phosphorylated forms of Aurora kinases. Interestingly, increased AKT phosphorylation or PTEN loss at baseline were not significantly correlated to %TGI. These results provide important insights into biomarker development for PI3K inhibitors in TNBC.

Multiplex Immunofluorescence in Formalin-Fixed Paraffin-Embedded Tumor Tissue to Identify Single-Cell-Level PI3K Pathway Activation.

Identifying cancers with high PI3K pathway activity is critical for treatment selection and eligibility into clinical trials of PI3K inhibitors. Assessments of tumor signaling pathway activity need to consider intratumoral heterogeneity and multiple regulatory nodes. We established a novel, mechanistically informed approach to assessing tumor signaling pathways by quantifying single-cell-level multiplex immunofluorescence using custom algorithms. In a proof-of-concept study, we stained archival formalin-fixed, paraffin-embedded (FFPE) tissue from patients with primary prostate cancer in two prospective cohort studies, the Health Professionals Follow-up Study and the Physicians' Health Study. PTEN, stathmin, and phospho-S6 were quantified on 14 tissue microarrays as indicators of PI3K activation to derive cell-level PI3K scores. In 1,001 men, 988,254 tumor cells were assessed (median, 743 per tumor; interquartile range, 290-1,377). PI3K scores were higher in tumors with PTEN loss scored by a pathologist, higher Gleason grade, and a new, validated bulk PI3K transcriptional signature. Unsupervised machine-learning approaches resulted in similar clustering. Within-tumor heterogeneity in cell-level PI3K scores was high. During long-term follow-up (median, 15.3 years), rates of progression to metastases and death from prostate cancer were twice as high in the highest quartile of PI3K activation compared with the lowest quartile (hazard ratio, 2.04; 95% confidence interval, 1.13-3.68). Our novel pathway-focused approach to quantifying single-cell-level immunofluorescence in FFPE tissue identifies prostate tumors with PI3K pathway activation that are more aggressive and may respond to pathway inhibitors.

A Novel Orthotopic Patient-Derived Xenograft Model of Radiation-Induced Glioma Following Medulloblastoma.

Simple SummaryRadiation-induced glioma (RIG) is a highly aggressive brain cancer arising as a consequence of radiation therapy, for which there is currently no effective treatment. In order to test new drugs in the hope of finding more effective therapies, we need mouse models that faithfully replicate human RIG. Our laboratory collected tumour cells at autopsy from a paediatric patient with RIG following treatment for a different brain tumour. Using these cells, we created a mouse brain tumour model that retains all the characteristics and features of the original patient tumour from which it was derived. This unique model allowed us to study the progression of RIG in the brain, and to identify drugs that may be effective in the treatment of this disease. This mouse model will also allow us to test the efficacy of new treatments, with the hope of improving the prognosis for patients diagnosed with this disease.Radiation-induced glioma (RIG) is a highly aggressive brain cancer arising as a consequence of radiation therapy. We report a case of RIG that arose in the brain stem following treatment for paediatric medulloblastoma, and the development and characterisation of a matched orthotopic patient-derived xenograft (PDX) model (TK-RIG915). Patient and PDX tumours were analysed using DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. While initially thought to be a diffuse intrinsic pontine glioma (DIPG) based on disease location, results from methylation profiling and WGS were not consistent with this diagnosis. Furthermore, clustering analyses based on RNA expression suggested the tumours were distinct from primary DIPG. Additional gene expression analysis demonstrated concordance with a published RIG expression profile. Multiple genetic alterations that enhance PI3K/AKT and Ras/Raf/MEK/ERK signalling were discovered in TK-RIG915 including an activating mutation in PIK3CA, upregulation of PDGFRA and AKT2, inactivating mutations in NF1, and a gain-of-function mutation in PTPN11. Additionally, deletion of CDKN2A/B, increased IDH1 expression, and decreased ARID1A expression were observed. Detection of phosphorylated S6, 4EBP1 and ERK via immunohistochemistry confirmed PI3K pathway and ERK activation. Here, we report one of the first PDX models for RIG, which recapitulates the patient disease and is molecularly distinct from primary brain stem glioma. Genetic interrogation of this model has enabled the identification of potential therapeutic vulnerabilities in this currently incurable disease.

Abstract B31: Single-cell multiplex immunofluorescence of formalin-fixed, paraffin-embedded prostate cancer tissue identifies PI3K pathway activation in two prospective cohort studies

Abstract Background: The PI3K pathway is activated in a large proportion of solid cancers, including prostate cancer. Loss of its inhibitor PTEN is associated with higher risk of lethal prostate cancer. However, clinical trials of PI3K and Akt inhibitors have had mixed results in prostate cancer, likely because patients were not selected for PI3K pathway activation. Here, we established a novel assessment of PI3K pathway activity on a single-cell level. Methods: Formalin-fixed, paraffin-embedded tumor tissue from patients diagnosed with primary prostate cancer during prospective follow-up of the Health Professionals Follow-up Study (HPFS) and the Physicians' Health Study (PHS) underwent centralized re-review by genitourinary pathologists. Tissue microarrays (TMAs) were sequentially stained for PTEN, stathmin, and phospho-S6 (pS6) as indicators of PI3K activation, and for AMACR. Slides were scanned on Vectra 3 (PerkinElmer), followed by multispectral imaging analysis. Tumor cells were identified using histology-based machine learning and AMACR staining intensity. Software for visualization of histomorphology and marker colocalization was developed. Cell-level PI3K activation scores were defined using a predefined analytical approach and compared to a data-driven approach. An independent, genomically validated core-level immunohistochemistry for PTEN loss and an mRNA signature of 105 PI3K/Akt/mTOR target genes served for validation. Patients were followed prospectively for lethal disease (metastases and cancer-specific death). Odds ratios (OR) with 95% confidence intervals (CIs) were estimated using logistic regression. Results: In 1,103 patients (719 from HPFS, 384 from PHS), 1.05 million tumor cells were assessed on 14 TMAs (median, 669 tumor cells per patient; interquartile range, 208 to 1362). Visualizations revealed intratumoral heterogeneity in PI3K activation in some tumors. Predefined and data-driven approaches to quantifying PI3K activity had similar results, with cell-level PI3K scores being driven by higher stathmin and pS6 and lower cell-level PTEN intensity. PI3K scores were higher in tumors with higher Gleason grade (ptrend < 0.001), core-level PTEN loss (p < 0.001; n = 866), and a higher transcriptional signature (ptrend = 0.025; n = 295). Over a median follow-up of 12.5 years with 95 lethal events, tumors in the highest quartile of PI3K activation were more than twice as likely to become lethal (OR, 2.56; 95% CI, 1.40–4.66; ptrend < 0.001) than those in the lowest quartile, even after adjusting for core-level PTEN status and Gleason grade (OR, 2.27; 95% CI, 1.01–5.08). Conclusion: Single-cell multiplex assessment of immunofluorescence PI3K markers can validly identify tumors with high activity of the PI3K pathway and may harbor more information than PTEN status alone. It provides a scalable measure that can be applied to routine specimens, including archival tissue from observational studies, and that can be used for patient selection into clinical trials of PI3K inhibitors. Citation Format: Konrad H. Stopsack, Ying Huang, Svitlana Tyekucheva, Travis A. Gerke, Clyde Bango, Habiba Elfandy, Xueliang Gao, Zhenying Cai, Thomas M. Roberts, Philip W. Kantoff, Lorelei A. Mucci, Massimo Loda. Single-cell multiplex immunofluorescence of formalin-fixed, paraffin-embedded prostate cancer tissue identifies PI3K pathway activation in two prospective cohort studies [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B31.

Abstract B18: Single-arm, open-label, phase II study of LY3023414 for the treatment of recurrent or persistent endometrial cancer

Abstract Background: Activating mutations of the PI3K/mTOR pathway are highly prevalent in endometrial cancers (EC), but monotherapy targets of this pathway have shown modest activity and are associated with poor tolerance. LY3023414 is a selective inhibitor of class I PI3K isoforms, mTOR, and DNA-PK. Building on phase I experience of LY3023414, we sought to evaluate the efficacy and safety of LY3023414 in PI3K pathway activated advanced EC. Methods: Eligible patients (pts) had PI3K pathway activation defined as (1) loss of PTEN function including whole or partial gene deletion, frame shift mutations, or non-sense mutations (PTEN missense mutations excluded) or (2) activating mutation in PIK3CA, AKT1, PIK3R1, PIK3R2, or mTOR. Up to 4 prior lines of therapy were allowed. Each cycle was 21 days. Efficacy assessments were performed every 2 cycles and treatment-related adverse events (AEs) were assessed per CTCAE v. 4.03. The primary objective was best overall response rate by RECIST 1.1. Results: Of the 28 pts enrolled, 25 (89%) had 1-2 lines of therapy and 3 pts (11%) had 3-4 prior lines of therapy. LY3023414 was dosed at the RP2D of 200 mg orally twice daily. Three pts were excluded from efficacy assessment due to toxicity, noncompliance or early patient withdrawal. The clinical benefit rate (CBR), defined as the percentage of pts with complete response (CR) + partial response (PR) + stable disease (SD) ≥12 weeks, was 56% (14/25). Five patients achieved PR; 9 pts achieved SD at best response. Three of the 5 PRs were sustained from 44-52 weeks; 1 pt with PR is still on therapy > 84 weeks. The median PFS and median OS were 2.53 (CI 1.18-2.99) months and 9.24 (CI 5-NR) months, respectively. Twenty-one pts underwent further genomic sequencing; 16 pts (76%) had a PIK3CA mutation, 6 pts (29%) had a PIK3R1 and 10 pts (48%) had a PTEN mutation. There were no significant co-mutations identified and a correlation of molecular alterations with response was not identified. Across all cycles, the most common grade 3 LY3023414-related adverse events (AEs) were lymphopenia (n=10), hypokalemia (n=5), hypophosphatemia (n=4), hyponatremia (n=3), and anemia (n=3). There were 3 LY3023414-related grade 4 AEs: allergic reaction (n=1), CPK rise (n=1) and hyponatremia (n=1). There were no LY3023414-related G5 AEs. Conclusions: In PI3K pathway-altered, heavily pretreated advanced EC, LY3023414 demonstrated a manageable safety profile and promising single-agent efficacy. Enrollment is now complete and updated response data will be presented. Citation Format: Maria M. Rubinstein, David Hyman, Imogen Caird, Roisin O'Cearbhaill, Rachel Grisham, William Tew, Martee Hensley, Paul Sabbatini, Carol Aghajanian, Alexia Iasonos, Helen Won, Duygu Selcuklu, Catherine Zimel, Vicky Makker. Single-arm, open-label, phase II study of LY3023414 for the treatment of recurrent or persistent endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B18.

Abstract B21: Protein quantitation assays for Akt, PI3K p110α, and PTEN to assess PI3K pathway activity in tumor tissue

Abstract Introduction: Many clinical trials for inhibitors targeting the PI3K pathway now include genetic screening of tumors to try to select probable treatment responders. Genetic testing identifies some relevant mutations, but this approach does not address many mechanisms of PI3K pathway regulation. Epigenetic changes and post-translational modifications may increase pathway activity without any detectable DNA mutation. Where mutations are detected, mutated proteins may not be expressed or may be quickly degraded. Direct measurement of oncogenic proteins may therefore offer more useful molecular profiles for characterizing cancer-driving alterations and matching patients with appropriate treatments. Purpose: The purpose of this study was to develop and implement protein quantitation assays to accurately assess PI3K pathway activity in tumors, as a means of matching patients with targeted cancer treatments. Methods: Protein extracted from tumor tissue lysate is subjected to proteolytic digestion to generate proteotypic peptides used for protein quantitation. Synthetic stable isotope-labeled peptides are added to the digest as an internal standard, after which the target peptides are immuno-enriched with antibodies coupled to magnetic beads. MS analysis is then performed by either eluting peptides for measurement with liquid chromatography multiple reaction monitoring tandem MS (LC-MRM-MS/MS) or by spotting beads onto a plate for matrix-assisted laser desorption MS (MALDI-MS). External calibration curves are used for accurate quantitation. Results: We developed and implemented MS-based methods to determine the concentration of PI3K pathway proteins in tumor samples. The developed methods enable precise and reproducible quantitation of Akt1, Akt2, PI3K P110α, and PTEN. We were able to distinguish and quantify specific isoforms, including phospho-Akt1 (Ser473), phospho-Akt2 (Ser474), and mutant-Akt (E17K). Endogenous protein levels were quantified in cell lines, fresh-frozen tumor samples, and formalin-fixed, paraffin embedded tumor tissue. 10 μg of protein extract per analyte was sufficient for quantitation. Subsets of assays could be multiplexed through simultaneous or sequential enrichment without compromising assay performance. Novel Aspect: The multiplexing of immuno-MALDI assays through sequential enrichment and the immuno-MRM-MS assays for PTEN and mutant Akt represent new developments. Recent adoption of monoclonal antibodies for Akt1 will facilitate this assay's translation to a clinical setting. Conclusions: This panel of quantitative proteomics assays enables comprehensive PI3K pathway assessment in tumor tissues. Previously collected genetically screened tumor samples were obtained with patient consent (as approved by the Jewish General Hospital Research Ethics Committee). Samples will be analyzed to assess PI3K pathway activity and determine its correlation to clinical features and response to PI3K-targeted treatments. Note: This abstract was not presented at the conference. Citation Format: Constance A. Sobsey, Robert Popp, Sahar Ibrahim, Bjoern C Froehlich, Adriana Aguilar-Mahecha, Mark Basik, Gerald Batist, Christoph Borchers. Protein quantitation assays for Akt, PI3K p110α, and PTEN to assess PI3K pathway activity in tumor tissue [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B21.

Abstract IA16: Role of PI3K signaling in T cell-mediated immune responses

Abstract A number of receptors are capable of activating the PI3K pathway in T cells, most prominently the T-cell receptor for antigen (TCR), the costimulatory receptor CD28, and the interleukin 2 receptor (IL-2R). PI3K signaling is critical for the initiation of effector T-cell responses, and activated multiple central downstream cascades including mTOR. Consistent with this, drugs that target mTOR, such as rapamycin, are effective inhibitors of T cell-mediated immune responses. Despite the important role of PI3K in T-cell activation, mTOR inhibition has been reported to “spare” regulatory T-cell (Treg) responses. This was of particular interest as Tregs, in contrast to all other T cells, constitutively express the high-affinity IL-2R, and thus should be poised for continual PI3K activation. Tregs, defined as cells that express the lineage-defining transcription factor Foxp3, are required for normal immune homeostasis. Foxp3-deficient mice (scurfy) and humans (patients with IPEX) are characterized by widespread and severe multiorgan autoimmunity and inflammation. As well, deletion of Tregs in adult mice leads to rapid and fulminant autoimmunity and death. In a series of studies conducted by members of my group over the past several years, as well as a variety of other laboratories, we have gained insight into the role of PI3K signals in Tregs, and a complex picture has emerged. We have observed that signals that elicit PI3K pathway activation in stimulated T cells, such as the cytokine IL-2, fail to do so in Tregs, despite the fact that Tregs express the IL-2R. Moreover, we have found that this is a selective “defect” in IL-2R signaling as the other major pathway linked to this receptor, STAT5, is intact. Specific loss of PI3K signaling in Tregs can be linked to expression of the lipid phosphatase PTEN. All T cells express PTEN in their resting state. However, while “conventional” T cells (i.e., non-Tregs) downregulate PTEN expression upon activation, Tregs uniquely do not. To test the importance of maintained PTEN expression in Tregs, we created mice with constitutive and with inducible deletion of PTEN specifically in Foxp3+ Tregs. These mice developed a dramatic phenotype of lupus-like autoimmunity, despite the presence of large numbers of Tregs, indeed more than in control mice. Moreover, we observed that loss of PTEN was followed by lineage instability, i.e., loss of Foxp3, and expression of proinflammatory cytokines such as IL-17 and IFNg. PI3K signals activate pathways important for T-cell metabolism, including c-myc, HIF-1a, and mTOR, and promote aerobic glycolysis. In contrast to most activated T cells, Tregs have been reported to be more reliant on fatty acid oxidation for their energy demands, and indeed loss of PTEN in Tregs dramatically augments aerobic glycolysis in these cells. However, most of the studies of Treg metabolism have focused on cells that were differentiated to become Tregs in vitro. In this system, naïve T cells are activated in the presence of the anti-inflammatory cytokine TGF-b, which induces them to express Foxp3. Such cells, “iTregs,” are easy to generate in large numbers and believed to be induced in vivo when antigen is encountered in noninflammatory settings in visceral organs and tissues. However, most T cells in lymphoid organs are thought to have originated in the thymus, are called tTregs, and are important for the prevention of immune responses to self-antigens. We have recently characterized the metabolism of these two Treg subsets. While, as reported, iTregs make minimal use of glycolysis, tTregs are in fact as glycolytic as effector T cells. We have further found that exposure of tTregs to TGF-b represses PI3K-mediated mTOR signaling, inhibits glucose transporter expression, and reprograms their metabolism to favor oxidative phosphorylation. Conversely, replicating the effects of inflammation via elevation of PI3K signaling has minimal effects on tTregs but dramatically enhances the glycolysis of normally oxidative iTregs, resulting in reduction of Foxp3 expression. Collectively, these findings suggest unique signaling and metabolic demands of Tregs and Treg subsets that may be leveraged in the design of therapeutics, both in vivo agents and adoptive cellular therapies. Citation Format: Laurence Turka. Role of PI3K signaling in T cell-mediated immune responses [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA16.

Negative cross talk between LIMK2 and PTEN promotes castration resistant prostate cancer pathogenesis in cells and in vivo.

Androgen deprivation therapy (ADT) and androgen receptor (AR) signaling inhibitors are front-line treatments for highly aggressive prostate cancer. However, prolonged inhibition of AR triggers a compensatory activation of PI3K pathway, most often due to the genomic loss of tumor suppressor PTEN, driving progression to the castration-resistant prostate cancer (CRPC) stage, which has very poor prognosis. We uncovered a novel mechanism of PTEN downregulation triggered by LIMK2, which contributes significantly to CRPC pathogenesis. LIMK2 is a CRPC-specific target. Its depletion fully reverses tumorigenesis in vivo. LIMK2 phosphorylates PTEN at five sites, degrading and inhibiting its activity, thereby driving highly aggressive oncogenic phenotypes in cells and in vivo. PTEN also degrades LIMK2 in a feedback loop, which was confirmed in prostates from PTEN-/- and PTEN+/+ mice. LIMK2 is also the missing link between hypoxia and PTEN degradation in CRPC. This is the first study to show a feedback loop between PTEN and its regulator. Uncovering the LIMK2-PTEN loop provides a powerful therapeutic opportunity to retain the activity and stability of PTEN protein by inhibiting LIMK2, thereby halting the progression to CRPC, ADT-resistance and drug-resistance.

Therapeutic Targeting of Signaling Pathways Related to Cancer Stemness.

The theory of cancer stem cells (CSCs) proposes that the different cells within a tumor, as well as metastasis deriving from it, are originated from a single subpopulation of cells with self-renewal and differentiation capacities. These cancer stem cells are supposed to be critical for tumor expansion and metastasis, tumor relapse and resistance to conventional therapies, such as chemo- and radiotherapy. The acquisition of these abilities has been attributed to the activation of alternative pathways, for instance, WNT, NOTCH, SHH, PI3K, Hippo, or NF-κB pathways, that regulate detoxification mechanisms; increase the metabolic rate; induce resistance to apoptotic, autophagic, and senescence pathways; promote the overexpression of drug transporter proteins; and activate specific stem cell transcription factors. The elimination of CSCs is an important goal in cancer therapeutic approaches because it could decrease relapses and metastatic dissemination, which are main causes of mortality in oncology patients. In this work, we discuss the role of these signaling pathways in CSCs along with their therapeutic potential.

Expression and Role of the G Protein-Coupled Estrogen Receptor (GPR30/GPER) in the Development and Immune Response in Female Reproductive Cancers.

Cancer is a major public health issue and represents the second leading cause of death in women worldwide, as female reproductive-related neoplasms are the main cause of incidence and mortality. Female reproductive cancers have a close relationship to estrogens, the principal female sex steroid hormones. Estrogens exert their actions by the nuclear estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). ERα, and ERβ act as transcription factors mediating genomic effects. Besides, the G protein-coupled estrogen receptor (GPER, formerly known as GPR30) was recently described as a seven-transmembrane receptor that mediates non-genomic estrogenic signaling, including calcium mobilization, cAMP synthesis, cleavage of matrix metalloproteinases, transactivation of epidermal growth factor receptor (EGFR), and the subsequent activation of PI3K and MAPK signaling pathways, which are the reasons why it is related to cellular processes, such as cell-cycle progression, cellular proliferation, differentiation, apoptosis, migration, and invasion. Since its discovery, selective agonists and antagonists have been found and developed. GPER has been implicated in a variety of hormone-responsiveness tumors, such as breast, endometrial, ovarian, cervical, prostate, and testicular cancer as well as lung, hepatic, thyroid, colorectal, and adrenocortical cancers. Nevertheless, GPER actions in cancer are still debatable due to the conflicting information that has been reported to date, since many reports indicate that activation of this receptor can modulate carcinogenesis. In contrast, many others show that its activation inhibits tumor activity. Besides, estrogens play an essential role in the regulation of the immune system, but little information exists about the role of GPER activation on its modulation within cancer context. This review focuses on the role that the stimulation of GPER plays in female reproductive neoplasms, specifically breast, endometrial, ovarian, and cervical cancers, in its tumor activity and immune response regulation.

Stimulation of Peroxisome Proliferator-Activated Receptor-Gamma (PPARγ) using Pioglitazone Decreases the Survival of Acute Promyelocytic Leukemia Cells through Up-Regulation of PTEN Expression.

The intertwining between cancer pathogenesis and aberrant expression of either oncogenes or tumor suppressor proteins ushered the cancer therapeutic approaches into a limitless road of modern therapies. For the nonce and among the plethora of promising anticancer agents, intense interest has focused on pioglitazone, a first in-class of Thiazolidinedione (TZD) drugs that is currently used to treat patients with diabetes. Intrigued by the overexpression of PPARγ in Acute Promylocytic Leukemia (APL), this study was designed to investigate the effects of pioglitazone in APL-derived NB4 cells. To assess the anti-leukemic effect of pioglitazone on myeloid leukemia cell lines, we used MTT and trypan blue assays. Given the higher expression level of PPARγ in NB4 cells, we then expanded our experiments on this cell line. To ascertain the molecular mechanism action of pioglitazone in APL-derived NB4 cells, we evaluated the expression levels of a large cohort of target genes responsible for the regulation of apoptosis, autophagy and cell proliferation. Afterward, to examine whether there is a correlation between PPARγ and the PI3K signaling pathway, the amount of Akt phosphorylation was evaluated using western blot analysis. Our results showed that pioglitazone exerted its cytotoxic effect in wild-type PTEN-expressing NB4 cells, but not in leukemic K562 cells harboring mutant PTEN; suggesting that probably this member of TZD drugs induced its anti-leukemic effects through a PTEN-mediated manner. Moreover, we found that not only pioglitazone reduced the survival rate of NB4 through the induction of p21-mediated G1 arrest, also elevated the intracellular level of Reactive Oxygen Species (ROS) which was coupled with upregulated FOXO3a. Notably, this study proposed for the first time that the stimulation of autophagy as a result of the compensatory activation of PI3K pathway may act as a plausible mechanism through which the anti-leukemic effect of pioglitazone may be attenuated; suggestive of the application of either PI3K or autophagy inhibitors along with pioglitazone in APL. By suggesting a mechanistic pathway, the results of the present study shed more light on the favorable anti-leukemic effect of pioglitazone and suggest it as a promising drug that should be clinically investigated in APL patients.

Abstract 4261: Development of quantitative multi-platform tests for easy readout of AR, ER, PI3K and MAPK pathway activity to unravel pathophysiology across cancer and tissue types

Abstract Introduction: Currently, the selection of targeted therapy is commonly based on genetic data, such as well-known gene mutations, but these mutations do not provide conclusive evidence for the functional activation of the affected pathway. For this reason, we have developed computational models to assess the molecular phenotype of individual tumors, determining the functional activity of oncogenic signaling pathways based on mRNA expression levels of the direct target genes of the respective pathway transcription factors (Verhaegh et al., Cancer Res 2014; Van Ooijen et al., Am J Pathol 2018; Van de Stolpe et al., Sci Rep 2019). For a broader application of these models, we translated them to RT-qPCR, RNA sequencing and microarray tests, for a quantitative and reproducible assessment of signaling pathway activity. Materials and methods: Focusing on the AR, ER, PI3K-FOXO and MAPK-AP1 pathways, we developed PCR primers and probes to assess mRNA expression levels of the pathways' direct target genes. For model parameter calibration, we performed controlled cell line experiments in which the respective pathways were inactivated and activated, to have calibration samples with known pathway activity status. Per pathway, the same set of calibration samples and the same set of target genes were used to develop RT-qPCR, RNA sequencing and microarray tests, to maximize similarity. The resulting OncoSignal tests can be used for a quantitative assessment of pathway activity on individual samples, giving a score on a 0-100 scale. We verified these tests on independent samples with known pathway status. We ran repeat experiments to assess the reproducibility within each platform, and tested the same samples on all three platforms to assess cross-platform concordance. Results: Using the OncoSignal score, low activity test samples were clearly separated from highly active ones. For example, AR-PCR scores went from 19±3 on androgen-deprived LNCaP cell lines to 70±2 after stimulation with DHT, and went down again to 33±1 and 23±1 when subsequently treated with bicalutamide and enzalutamide, respectively. PI3K-FOXO-microarray scores went from 12±1 on control MCF7 experiments to 50±4 after alpelisib treatment. Measurements on 100+ samples on all three platforms showed Pearson correlations between 0.93 and 0.99 in OncoSignal scores, with absolute differences typically below 10. Conclusion: We developed broadly applicable tests to assess AR, ER, PI3K-FOXO and MAPK-AP1 pathway activity in a quantitative manner on individual samples, with good reproducibility on repeat experiments and good concordance between PCR, RNA sequencing and microarray measurements. These tests can be used to unravel disease pathophysiology in (pre-)clinical samples. The added value of OncoSignal for therapy response prediction is currently being evaluated in different studies. Citation Format: Martijn Akse, Henk van Ooijen, Anke Pierik, Sandra van den Bosch, Hans van Zon, Laurent Holtzer, Anne van Brussel, Márcia Inda, Yvonne Wesseling-Rozendaal, Wim Verhaegh, Eveline den Biezen-Timmermans. Development of quantitative multi-platform tests for easy readout of AR, ER, PI3K and MAPK pathway activity to unravel pathophysiology across cancer and tissue types [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4261.

Abstract 632: Novel BRAF gene fusions in pediatric histiocytic neoplasms respond differentially to RAF targeted therapies based on dimerization profiles

Abstract Introduction: Pediatric histiocytic neoplasms are clonal hematopoietic disorders driven by mutations activating the mitogen-activated protein kinase (MAPK) pathway, such as BRAF- V600E. In non-BRAFV600E cases, we investigated alternative MAPK mutations and found two novel BRAF gene fusions. We have previously shown effective targeting of BRAF fusions (KIAA1549-BRAF) in pediatric low-grade gliomas (PLGGs) with paradox breaker RAF inhibitor (RAFi), PLX8394 and MEK inhibitors (MEKi). Here, we investigate the distinct responsiveness of novel BRAF fusions to RAFi therapies and explore the mechanistic basis of such differential responses compared to other BRAF fusions. Method: Two histiocytic patient tumors were analyzed using the CHOP Comprehensive Next- Gen Sequencing Solid Tumor Panel and a targeted RNA-seq panel for 106 fusion partner genes. Novel BRAF fusions identified were sub-cloned to create heterologous cell models. Immunoblotting of serum starved cells measured MAPK and PI3K pathway activation and soft agar assay tested for oncogenic phenotypes driven by BRAF fusions, along with response to PLX8394, LY3009120 (pan-RAF dimer inhibitor), and MEKi. Co-immunoprecipitation assays using Myc- and Flag-tagged BRAF fusion constructs assessed dimerization profiles, with or without inhibitors. Result: In the two M- and L-type histiocytic neoplasms assessed, we found novel and rare BRAF gene fusions, MTAP-BRAF and MS4A6A-BRAF, respectively. Both BRAF fusions activated the MAPK/ PI3K pathways and showed homo- and hetero-dimerization with BRAF and the respective N-terminal fusion partner. Compared to common BRAF fusions, MTAP-BRAF and MS4A6A- BRAF did not respond to PLX8394 due to no disruption of active fusion homo- and hetero-dimers, which was in turn was due to the untargeted, stable dimerization mediated by the N-terminal fusion partners. Conversely, we observed robust suppression with LY3009120 that bound fusion dimers and kept them in an inactivate confirmation. MEKi selumetinib and trametinib also suppressed fusion driven signaling and oncogenic phenotypes. Conclusion: We show that novel MTAP-BRAF and MS4A6A-BRAF fusions, found in histiocytic tumors, do not respond to RAFi PLX8394 but are targeted by LY3009120 or MEKi. Our finding that PLX8394 does not disrupt MTAP-BRAF or MS4A6A-BRAF dimerization due to contribution of N-terminal partners defines a novel paradigm for the distinct mechanisms sought by BRAF fusions in response to RAFi therapy. We show potent suppression of these mechanistically distinct BRAF fusions with MEKi or LY3009120 which function independent of vulnerability to fusion dimerization. Overall, this study highlights the unique and differential biology hijacked by BRAF fusions in response to RAFi and further warrants detailed mechanistic classification of BRAF fusions based on their responsiveness to targeted agents. Citation Format: Payal Jain, Lea F. Surrey, Joshua Straka, Pierre Russo, Richard Womer, Marilyn Li, Phillip B. Storm, Angela J. Waanders, Michael Hogarty, Jennifer Picarsic, Adam C. Resnick. Novel BRAF gene fusions in pediatric histiocytic neoplasms respond differentially to RAF targeted therapies based on dimerization profiles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 632.

Abstract 661: Synergistic antitumor activity of futibatinib, an FGFR1-4 inhibitor, and TAS-117, a selective AKT inhibitor, in FGFR-deregulated cancer models

Abstract Background: TAS-117 is a potent oral allosteric non-ATP inhibitor selective for AKT. A phase 1 study with TAS-117 is ongoing in patients with endometrial cancer and ovarian clear cell carcinoma. Futibatinib (TAS-120) is a selective, irreversible small-molecule fibroblast growth factor receptor (FGFR) 1-4 inhibitor, with potent antiproliferative activity in preclinical models with FGFR aberrations. Phase 2 studies are ongoing in patients with various FGFR-deregulated cancers. We evaluated the antitumor activity of futibatinib plus TAS-117 in FGFR-aberrant cancer models. Methods: Effects of futibatinib plus TAS-117 were investigated in human endometrial FGFR2-N549K-mutated AN3CA cancer cells and small-cell lung cancer FGFR1-amplified DMS114 cells. Activation of RAS and PI3K pathways was assessed by detecting phosphorylation of ERK1/2, AKT, and S6. Antitumor combination indices (CI) for cell viability over time were determined with the Chou-Talalay median-effect method and CalcuSyn software; a CI<0.9 indicates synergy. Antitumor effects of futibatinib (15 mg/kg/d) plus TAS-117 (4 or 8 mg/kg/d) or monotherapy were also evaluated in a mouse AN3CA xenograft model. FGFR signaling, apoptosis (via caspase activity and PARP cleavage), and relative tumor volume (RTV; compared with Day 0) were assessed. Results: Futibatinib and TAS-117 synergistically increased cell death and decreased cell growth in AN3CA (CI<0.40) and DMS114 (CI<0.40) cells, respectively. Apoptosis induction was readily detected with futibatinib plus TAS-117 in AN3CA cells, but low activity was observed with monotherapy. In both AN3CA and DMS114 cells, futibatinib suppressed ERK1/2, but not AKT phosphorylation; the futibatinib plus TAS-117 combination suppressed phosphorylation of both ERK1/2 and AKT. In AN3CA xenografts, combination therapy inhibited the FGFR, PI3K, and MEK pathways. Futibatinib plus TAS-117 increased PARP cleavage more than monotherapy. Compared with Day 0, RTV on Day 15 was 60.71 in the control, 13.40 with futibatinib monotherapy, and 33.63 with TAS-117 (8 mg/kg/d) monotherapy. Treatment with 3 different futibatinib plus TAS-117 combination dosing regimens resulted in marked reductions in RTVs on Day 15 (2.16, 0.53, and 0.22). Conclusions: Our findings suggest that futibatinib plus TAS-117 has synergistic antitumor effects in FGFR-aberrant tumors. A phase 1/2 study of this combination in advanced solid tumors (JapicCTI-194864) is ongoing. Citation Format: Junya Iwasaki, Takuya Kuramoto, Toshiharu Komori, Hitoshi Saito, Hiroshi Hirai. Synergistic antitumor activity of futibatinib, an FGFR1-4 inhibitor, and TAS-117, a selective AKT inhibitor, in FGFR-deregulated cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 661.

Abstract 663: The dual PI3K-δ/PI3K-γ inhibitor duvelisib inhibits signaling and proliferation of solid tumor cells expressing PI3K-δ and/or PI3K-γ

Abstract Duvelisib is an oral, dual PI3K-δ,γ inhibitor that is FDA-approved for treatment of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and follicular lymphoma (FL) following ≥2 prior systemic therapies. In addition to targeting malignant B cells, duvelisib regulates key non-malignant immune cells in the tumor microenvironment (TME), including Tregs and immunosuppressive myeloid cells. However, direct effects of duvelisib on solid tumor cells have not been tested. Here, we used the GEPIA webserver to analyze the expression of PIK3CD [PI3K-δ] and PIK3CG [PI3K-γ] from the TCGA database. Whereas PIK3CG was highly expressed in hematological malignancies (log2 median transcript per million, TPM+1 > 2) but not in solid tumors, PIK3CD was highly expressed in hematological malignancies (log2TPM+1 = 5.1 in DLBCL) as well as in a number of solid tumors, including melanoma (log2TPM+1 = 4.4) and head and neck squamous cell carcinoma (HNSCC) (log2TPM+1 = 3.2). Further, PIK3CD was more highly expressed in melanoma and HNSCC human tumor samples compared to adjacent normal tissue, suggesting a potential tumorigenic role of PI3K-δ. We also checked the expression of PIK3CD in human tumor cell lines from the Cancer Cell Line Encyclopedia database and found that 57% and 22% of the melanoma and HNSCC cell lines, respectively, expressed high levels (log2TPM+1 > 2) of PIK3CD, while 7% of melanoma cell lines expressed high levels of PIK3CG. To assess possible direct effects of duvelisib on solid tumor cells, the growth inhibitory activity of duvelisib was evaluated across 7 melanoma and 3 HNSCC cell lines expressing high levels of PI3K-δ and/or PI3K-γ. Cell viability was measured after 72-hour treatment with duvelisib (0.5nM-10μM) and reported as growth inhibition (GI) at 1μM. Duvelisib showed strong anti-proliferative activity (50-100% GI) in 3 out of 7 melanoma cell lines and 1 out of 3 HNSCC cell lines, and moderate activity (15-50% GI) in 3 melanoma cell lines and an additional HNSCC cell line. Duvelisib inhibited PI3K pathway activity, as measured by p-AKT and c-myc, in all the cell lines in which strong anti-proliferative activity was observed. Taken together, these findings indicate that in addition to the established effects of duvelisib on malignant B cells and non-malignant immune cells of the TME, duvelisib can also directly inhibit signaling and proliferation of solid tumor cells expressing PI3K-δ and/or PI3K-γ, providing further rationale for clinical investigation of duvelisib for the treatment of solid tumors. Accordingly, the combination of duvelisib with pembrolizumab is currently being evaluated in patients with head and neck squamous cell carcinoma. Citation Format: SILVIA COMA, David T. Weaver, Jonathan A. Pachter. The dual PI3K-δ/PI3K-γ inhibitor duvelisib inhibits signaling and proliferation of solid tumor cells expressing PI3K-δ and/or PI3K-γ [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 663.