Phosphatidylinositol 3-kinase Pathway Research Articles

Targeting the PI3K/AKT/mTOR Pathway in Hormone-Positive Breast Cancer.

Approximately 70% of invasive breast cancers have some degree of dependence on the estrogen hormone for cell proliferation and growth. These tumors have estrogen and/or progesterone receptors (ER/PR+), generally referred to as hormone receptor positive (HR+) tumors, as indicated by the presence of positive staining and varying intensity levels of estrogen and/or progesterone receptors on immunohistochemistry. Therapies that inhibit ER signaling pathways, such as aromatase inhibitors (letrozole, anastrozole, exemestane), selective ER modulators (tamoxifen), and ER down-regulators (fulvestrant), are the mainstays of treatment for hormone-receptor-positive breast cancers. However, de novo or acquired resistance to ER targeted therapies is present in many tumors, leading to disease progression. The PI3K/AKT/mTOR pathway is implicated in sustaining endocrine resistance and has become the target of many new drugs for ER+ breast cancer. This article reviews the function of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway and the various classes of PI3K pathway inhibitors that have been developed to disrupt this pathway signaling for the treatment of hormone-receptor-positive breast cancer.

Anthrax lethal factor cleaves regulatory subunits of phosphoinositide-3 kinase to contribute to toxin lethality.

Anthrax lethal toxin (LT), produced by Bacillus anthracis, comprises a receptor-binding moiety, protective antigen and the lethal factor (LF) protease1,2. Although LF is known to cleave mitogen-activated protein kinase kinases (MEKs/MKKs) and some variants of the NLRP1 inflammasome sensor, targeting of these pathways does not explain the lethality of anthrax toxin1,2. Here we report that the regulatory subunits of phosphoinositide-3 kinase (PI3K)-p85α (PIK3R1) and p85β (PIK3R2)3,4-are substrates of LF. Cleavage of these proteins in a proline-rich region between their N-terminal Src homology and Bcr homology domains disrupts homodimer formation and impacts PI3K signalling. Mice carrying a mutated p85α that cannot be cleaved by LF show a greater resistance to anthrax toxin challenge. The LF(W271A) mutant cleaves p85α with lower efficiency and is non-toxic to mice but can regain lethality when combined with PI3K pathway inhibitors. We provide evidence that LF targets two signalling pathways that are essential for growth and metabolism and that the disabling of both pathways is likely necessary for lethal anthrax infection.

3O Efficacy of the triple combination of Estrogen Receptor, CDK4/6 and PI3K pathway inhibitors in ex-vivo models of breast cancer

Activation of the Phosphatidylinositol 3-kinase (PI3K) pathway is common in Estrogen receptor (ER)-positive breast cancer (BC) and it has been associated with resistance to endocrine therapies (ET). The crosstalk between the Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K pathways represents a strong rationale for co-targeting both axes to inhibit tumor growth. Moreover, combined treatment with PI3K and CDK4/6 inhibitors has been shown to overcome CDK4/6 inhibitor resistance in BC cells. We investigated the triple combination of ER, CDK4/6, and PI3K pathway inhibitors in ex vivo BC models as a novel strategy to overcome endocrine and CDK4/6-resistance.

LBA18 Overall survival (os) results from SOLAR-1, a phase III study of alpelisib (ALP) + fulvestrant (FUL) for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC)

Phosphatidylinositol 3-kinase (PI3K) pathway hyperactivation due to PIK3CA mutations contributes to poor survival in patients (pts) with HR+, HER2– ABC (Mosele 2020). In the phase III SOLAR-1 trial of pts with progression on/after aromatase inhibitor (AI), the PI3Kα inhibitor ALP together with FUL significantly improved progression-free survival (PFS) in the PIK3CA-mutant (mut) cohort (HR 0.65; 95% CI 0.50-0.85; P<0.001; median [m] PFS 11.0 mo with ALP + FUL vs 5.7 mo with placebo [PBO] + FUL), a population with poor prognosis. At final PFS analysis, the first interim OS results were immature. Men/postmenopausal women (N=572) with HR+, HER2– ABC with no prior chemotherapy treatment (tx) and whose disease progressed on/after AI were randomized 1:1 to receive ALP (300 mg PO QD) or PBO, + FUL (500 mg IM on D1 and D15 of C1 then D1 of each 28-d cycle) and stratified by presence of lung and/or liver metastases and prior cyclin-dependent kinase 4/6 inhibitor. OS in the PIK3CA-mut cohort, a key secondary endpoint, was evaluated by Kaplan-Meier methodology; a 1-sided stratified log-rank test was performed with an O’Brien-Fleming efficacy boundary of P≤0.0161. With mOS follow-up of 30.8 mo, mOS was 39.3 mo (95% CI 34.1-44.9) with ALP + FUL and 31.4 mo (95% CI 26.8-41.3) with PBO + FUL (HR 0.86; 95% CI 0.64-1.15; P=0.15); OS did not cross the prespecified boundary. Median time to chemotherapy (TTC) was 23.3 mo (95% CI 15.2-28.4) with ALP + FUL and 14.8 mo (95% CI 10.5-22.6) with PBO + FUL (HR 0.72; 95% CI 0.54-0.95). In pts with lung and/or liver metastases, mOS was 37.2 mo (95% CI 28.7-43.6) with ALP + FUL and 22.8 mo (95% 19.0-26.8) with PBO + FUL (HR 0.68; 95% CI 0.46-1.00). No new safety signals were observed with longer follow-up. Though not statistically significant, OS was prolonged by a clinically relevant ≈8 mo with ALP when added to FUL in HR+, HER2−, PIK3CA-mut ABC. mTTC was also prolonged with ALP + FUL vs PBO + FUL. Coupled with the statistically and clinically significant PFS, these data further support ALP + FUL in this poorer prognostic population of pts with PIK3CA-mut ABC.

LFA-1 signals to promote actin polymerization and upstream migration in T cells.

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the phosphoinositide 3-kinase (PI3K) and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, whereas VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl (also known as CBL), failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior.This article has an associated First Person interview with the first author of the paper.

Prevalence of Phosphatidylinositol-3-Kinase (PI3K) Pathway Alterations and Co-alteration of Other Molecular Markers in Breast Cancer.

Background: PI3K/AKT signaling pathway is activated in breast cancer and associated with cell survival. We explored the prevalence of PI3K pathway alterations and co-expression with other markers in breast cancer subtypes.Methods: Samples of non-matched primary and metastatic breast cancer submitted to a CLIA-certified genomics laboratory were molecularly profiled to identify pathogenic or presumed pathogenic mutations in the PIK3CA-AKT1-PTEN pathway using next generation sequencing. Cases with loss of PTEN by IHC were also included. The frequency of co-alterations was examined, including DNA damage response pathways and markers of response to immuno-oncology agents.Results: Of 4,895 tumors profiled, 3,558 (72.7%) had at least one alteration in the PIK3CA-AKT1-PTEN pathway: 1,472 (30.1%) harbored a PIK3CA mutation, 174 (3.6%) an AKT1 mutation, 2,682 (54.8%) had PTEN alterations (PTEN mutation in 7.0% and/or PTEN loss by IHC in 51.4% of cases), 81 (1.7%) harbored a PIK3R1 mutation, and 4 (0.08%) a PIK3R2 mutation. Most of the cohort consisted of metastatic sites (n = 2974, 60.8%), with PIK3CA mutation frequency increased in metastatic (32.1%) compared to primary sites (26.9%), p < 0.001. Other PIK3CA mutations were identified in 388 (7.9%) specimens, classified as “off-label,” as they were not included in the FDA-approved companion test for PIK3CA mutations. Notable co-alterations included increased PD-L1 expression and high tumor mutational burden in PIK3CA-AKT1-PTEN mutated cohorts. Novel concurrent mutations were identified including CDH1 mutations.Conclusions: Findings from this cohort support further exploration of the clinical benefit of PI3K inhibitors for “off-label” PIK3CA mutations and combination strategies with potential clinical benefit for patients with breast cancer.

Abstract 621: Combined inhibition of checkpoint kinase 1 (CHK1) and phosphoinositide 3-kinase (PI3K) pathways induces greater replication stress and DNA damage in high-grade serous ovarian cancer (HGSOC)

Abstract HGSOC is characterized by universal TP53 mutations and consequent G1 cell cycle dysfunction, thus relies on ATR/CHK1-mediated G2/M arrest for survival. CHK1 is also necessary for chromosome stability by mitigating replication stress (RS) and promoting homologous recombination (HR) DNA repair following DNA damage. However, clinical responses to CHK1 inhibitor (CHK1i) monotherapy have been variable in HGSOC, requiring new treatment strategies. We thus conducted a high throughput drug combination screening of a CHK1i, prexasertib (Prex) with 1,912 drugs in HGSOC cell lines (BRCA wild-type [BRCAwt: OVCAR5 and OVCAR8] and BRCA2 mutant [BRCA2m: PEO1]). In this screen, 24 drugs showed synergistic cytotoxicity with Prex, including 14 (58%) chemotherapies, 6 (25%) PI3K pathway inhibitors and 4 (17%) targeted drugs. We prioritized the PI3K pathway inhibitors given the PI3K pathway is activated in &amp;gt;45% of HGSOC and associated with increased cell survival, DNA repair and chemo-resistance. We confirmed the combination of Prex and a dual PI3K/mTOR inhibitor LY3023414 (LY302) yielded synergistic cytotoxicity (combination index&amp;lt;1) in a panel of HGSOC cell lines (OVCAR3, OVCAR5, OVCAR8, OV90 and PEO4 [all BRCA-proficient] and PEO1) by XTT and colony formation assays. OVCAR8 and PEO1 were used for further mechanistic studies. With clinically attainable concentrations of Prex (5 nM) and LY302 (200 nM), Prex+LY302 increased caspase 3/7 activation compared to Prex (increased 2,407% and 39%, respectively; P&amp;lt;0.01) or LY302 (increased 2,457% and 57%, respectively; P&amp;lt;0.001) in both cell lines. Prex+LY302 also enhanced the percentage of cells with ≥5 γH2AX foci relative to Prex (increased 39% and 22%, respectively; P&amp;lt; 0.001) or LY302 (increased 48% and 32%, respectively; P&amp;lt;0.001) in both cells, indicating greater DNA damage. We found Prex+LY302 induced a significant HR deficiency as manifested by decreased DR-GFP reporter activity compared to Prex (decreased 37% and 28%, respectively; P&amp;lt;0.01) or LY302 (decreased 44% and 63%, respectively; P&amp;lt;0.01), suggesting Prex+LY302 causes DNA damage likely via reducing HR functionality. Cell cycle analysis showed an imposed S phase arrest in cells treated with Prex+LY302 compared to those treated with Prex (increased 30% and 35%, respectively; P&amp;lt;0.05) or LY302 (increased 38% and 51%, respectively; P&amp;lt;0.01), implying Prex+LY302 increases RS or DNA damage during S phase. Supporting this notion, Prex+LY302 augmented RS as evidenced by increased phospho-RPA+/γH2AX+ populations compared with Prex (increased 37% and 38%, respectively; P&amp;lt;0.05) or LY302 (increased 80% and 79%, respectively; P&amp;lt;0.001). Overall, our results suggest that dual inhibition of CHK1 and PI3K pathways results in greater RS, DNA damage and subsequent cell death in HGSOC cells independent of BRCA mutation status. Citation Format: Tzu-Ting Huang, Jayakumar Nair, Ethan Brill, Xiaohu Zhang, Kelli Wilson, Lu Chen, Craig J. Thomas, Jung-Min Lee. Combined inhibition of checkpoint kinase 1 (CHK1) and phosphoinositide 3-kinase (PI3K) pathways induces greater replication stress and DNA damage in high-grade serous ovarian cancer (HGSOC) [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 621.

Abstract 1908: Kinome rewiring upon acquisition of resistance to PI3K inhibitors in ER+ breast cancer

Abstract Hyperactivation of the phosphatidylinositol-3 kinase (PI3K) pathway is a critical step in oncogenesis and plays a role in the development of treatment resistance in estrogen receptor-positive (ER+) breast cancer. Targeted inhibition of PI3K (PI3Ki) was recently approved in combination with endocrine therapy for advanced ER+ breast cancer. As most tumor-targeted therapies are encumbered by the development of secondary resistance, there remains a need to identify novel therapeutic strategies to prevent and overcome PI3Ki resistance. We treated mice bearing anti-estrogen-resistant ER+ breast cancer xenografts with the anti-estrogen fulvestrant +/- the pan-PI3Ki GDC-0941. The drug combination induced partial regression of tumors, followed by regrowth despite continued drug treatment. We harvested tumors before, during, and after acquisition of resistance to the drug combination and performed quantitative phosphoproteomic profiling. Using LC-MS/MS, we identified 203 phosphopeptides and 227 unique phosphorylation sites in the drug-resistant tumors, with clusters of 87 upregulated and 116 downregulated phosphopeptides. The NetPhorest analysis platform predicted hyperactivation of 6 kinases and hypoactivation of 16 kinases in drug-resistant tumors. Notably, two hyperactive kinases, MAPK1 and HIPK2, were predicted to target substrates MARKS (at Ser27 in the calcium binding domain) and NS1BP (at Ser322 in the homeodomain kelch repeat). MARCKS is a filamentous actin cross-linking protein, while the kelch repeats of NS1BP are known to associate with F-actin to stabilize actin filaments. In addition, the most significant predicted kinase among the cluster of 116 downregulated phosphopetides in the drug-resistant tumors was “PKCiota”, predicted to target PARD3 at Ser852. Previous work showed that PARD3 is central for the formation of tight junctions. These data collectively suggest a dynamic role of kinase-substrate interactions involved in resistance to combined anti-estrogen/PI3Ki treatment, including filamentation actin formation and stabilization, as well as the deregulation of tight junction formation. Citation Format: Jacqueline Reedy Griffin, Nicole Traphagen, Arminja Kettenbach, Todd Miller. Kinome rewiring upon acquisition of resistance to PI3K inhibitors in ER+ breast cancer [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 1908.

Abstract 97: Analysis of Pten-null mouse astrocytes revealed overlapping transcribed genes with human glioblastoma transcriptome

Abstract Glioblastoma is an aggressive form of brain cancer with universally poor prognosis. Even with optimal therapy, the cancer recurs frequently. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene whose mutations are often found in multiple human cancers. PTEN encodes a lipid phosphatase and serves as a negative regulator of the phosphoinositide 3-kinase (PI3K) pathway. Cumulative evidence suggests that PTEN mutations play significant roles in tumorigenesis of glioblastoma as 60% of glioblastoma patients acquired mutations in this gene. Based on our preliminary data, transcriptomic profiling of primary astrocytes derived from Pten knockout mice revealed an overlapping enrichment of mitotic regulators and cell cycle related pathways with human GBM. 4 genes (RCC1, RCC2, NEK2, and CHK2) were validated to be significantly upregulated in PTEN KO astrocytes by qRT-PCR. Regulator of chromatin condensation 2 (RCC2) has been reported to be guanine-nucleotide exchange factor (GEF) for Rac1 and RalA GTPase and act as a regulator of cell cycle progression. Accumulating evidence revealed a pro-tumorigenic role of RCC2 in multiple cancers such as tumor growth and tumor metastasis. Via siRNA inhibition of RCC2, we demonstrated that knocking down of RCC2 in glioblastoma cell lines suppresses cell growth and RCC2 is essential for both G1-S and G2-M progression. Additionally, RCC2 knocking down and a PI3K inhibitor LY294002 inhibit glioblastoma cell viability in a synergistic manner. In summary, PTEN deficiency leads to the upregulation of RCC2, which may promote tumorigenesis of glioblastoma by dysregulating cell cycle progression.To further explore the RCC2 roles, we will find the potential downstream target of RCC2 in dysregulating cell cycle and its potential therapeutic effect in vivo. Funding: General Research Fund Grant #14113519 from The Research Grants Council of Hong Kong Citation Format: Tian Liu, Penelope Mei-Yu Or, Chi-Wai Wong, Stanley Kwok-Kuen Cheung, Yubing Wang, Yiwei Wang, Andrew M. Chan. Analysis of Pten-null mouse astrocytes revealed overlapping transcribed genes with human glioblastoma transcriptome [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 97.

Abstract 1113: Evaluation of novel therapeutics using models from the Mayo Clinic GBM patient-derived xenograft (PDX) collection

Abstract The Mayo Clinic collection of patient-derived xenograft (PDX) models has been developed from the direct implantation of fresh glioblastoma samples into immunodeficient mice. Over 95 models have been extensively characterized with regards to histology, invasion, flank and orthotopic growth rates, and molecular profiling. Evaluation of orthotopic tumors demonstrated clear infiltration of tumor cells into the brain parenchymal in 90% of PDXs with contralateral hemisphere involvement in 64%. Extensive molecular profiling confirmed that our collection of PDXs captures the genetic heterogeneity of GBM, with the majority of known alterations represented at a frequency similar to the TCGA including gain of chromosome 7 and loss of chromosome 10. TERT promoter mutations were the most frequent overall (86%) followed by homozygous deletion of CDKN2A (70%). Multiple alterations were identified in the p53 pathway, including TP53 mutations (36%) and MDM2/4 (10%/2%) amplification. PI3-kinase pathway alterations mainly involved PTEN (48%) while MAP kinase pathway alterations were present in a subset of PDXs (NF1 alterations 17%, BRAF 4%). IDH mutations were found in only two models. Gene expression analysis revealed that our PDX collection includes all GBM subtypes with classical being the most represented (46% vs mesenchymal 31% and proneural 23%). MGMT promoter methylation was also observed in 45% of PDXs. The high fidelity of PDX models to their human counterpart makes these models ideal for testing standard of care and novel targeted therapies. To assess if PDXs are predictive of patient treatment response, 37 orthotopic PDX lines were treated with radiation (RT), temozolomide (TMZ), or concurrent RT/TMZ. Across all PDXs, RT and TMZ demonstrated median survival extension of 90 and 46 days relative to vehicle. Concurrent RT/TMZ further extended survival by 146 days. MGMT promoter methylation was strongly associated with response to TMZ-based strategies but not RT alone.This observation suggests that our PDX collection possesses clinically relevant predictive value. To date, we have characterized treatment responses to over 135 regimens including both GBM standards of care and targeted strategies in our PDX models. Recent lab efforts strive to define the effect of blood brain barrier disruption on drug delivery. With over 20 years of experience, our group has extensive expertise in the development of clinically relevant study designs focused on examining efficacy, tolerability, and pharmacokinetic/pharmacodynamic assessments. Since 2016, we have shared over 5200 individual samples (tumor tissue, cells, DNA/RNA, protein extracts, tissue microarray slides) in both internal and external collaborators. Our models have been utilized in over 41 NIH funded grants, including 33 funded RO1s, as well as by the pharmaceutical community with 50 working relationships to date (20 contracts, 30 collaborations). Finally, our PDX collection has been used in over 120 peer-reviewed published manuscripts. Citation Format: Danielle Burgenske, Ann C. Mladek, Rachael A. Vaubel, Shulan Tian, Mark A. Schroeder, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. Evaluation of novel therapeutics using models from the Mayo Clinic GBM patient-derived xenograft (PDX) collection [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 1113.

Abstract 5335: CA102N suppresses the growth of mouse colon cancer by inhibition of PI3K pathway and immune modulation

Abstract It is well documented that COX-2 inhibitors have the potential in preventing or treating colorectal cancer. However, the cardiovascular toxicity of COX-2 inhibitors hampered further development of these inhibitors in cancer management. CA102N is a covalently bound conjugate of the biological polymer sodium hyaluronate (NaHA) and nimesulide (Nim) aiming to deliver Nim directly to the tumor tissues to limit the systemic toxicity. Here, we report the antitumor effect of CA102N in colon cancer and the possible mechanism associated with its antitumor activity. The antitumor efficacy of CA102N was tested in the CT-26 syngeneic Balb/c mouse colon cancer model. The effect of CA102N on inflammation was examined by serum cytokines, intratumor and urinary COX-2 metabolites. Immune modulation was determined by profiling TILs with flow cytometry. Cell signaling proteins were measured by western blot analysis. CA102N (50 mg/kg and 200 mg/kg) i.v. injected mice carrying CT26 mouse colon tumor showed significantly reduced average tumor weight (726.1 ± 335.2 mg and 633.4 ± 453.5 mg, respectively) compared to that of the vehicle control treated mice (1392.0 ± 523.8, p &amp;lt; 0.05). The reduction of tumor growth by CA102N in this particular tumor model was comparable to the mice having received TAS102 (100 mg/kg) p.o. daily for two weeks. Intratumor COX-2 metabolite analysis showed that CA102N (200 mg/kg) reduced levels of both PGE2 and PGI2 by 31% and 58% (p&amp;lt; 0.05), respectively compared to that of control group. The levels of urinary COX-2 metabolites in CA102N treated CT26 tumor bearing mice were similar to that of control mice. Results of serum cytokine analysis suggest that levels of MCP-1 (109.9 ± 24.3 pg/ml) and IL6 (18.0 ± 3.8 pg/ml) in CA102N (50 mg/kg) treated mice were significantly lower than that of control group (175.4 ± 21.0 pg/ml) and (123.3 ± 55.8 pg/ml), respectively (p &amp;lt; 0.05). Furthermore, TILs profiling showed that FOXP3+ T-regulatory immune cell population was reduced by almost 63% in CA102N (50 mg/kg) treated mice whereas the number of CD8+ T cells in CA102N (50 mg/kg) treated tumors was about 2-fold higher than that of the control group. In contrast, CA102N (200 mg/kg) treatment neither altered the serum cytokines and nor increased CD8+ T-cell populations in the tumor. Additionally, the protein expression of pAkt and pS6 in CA102N (200 mg/kg) treated tumor tissues were significantly down-regulated by more than 50% compared to that of control group, while CA102N (50 mg/kg) treatment only slightly altered the abundance of these proteins, suggesting antitumor activity of CA102N in different doses can potentially be mediated by differential mechanisms. Together, these findings suggest that the lower dose of CA102N exerts antitumor activity in mouse colon tumor model by immune modulation whereas the antitumor activity of the higher dose of CA102N might be mediated through inhibition of PI3kinase pathway on tumor tissues. Citation Format: Peiying Yang, Tara Conway, Patrea Rhea, Bo Wei, Eskouhie Tchaparian. CA102N suppresses the growth of mouse colon cancer by inhibition of PI3K pathway and immune modulation [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 5335.

Protein Expression Analysis of an In Vitro Murine Model of Prostate Cancer Progression: Towards Identification of High-Potential Therapeutic Targets.

Background: Prostate cancer (PC) is the most frequently diagnosed cancer among men worldwide. The poor prognosis of PC is largely due to late diagnosis of the disease when it has progressed to advanced stages marked by androgen-independence. We interrogated proteomic signatures that embody the transition of PC from an androgen-dependent (AD) to an androgen-independent (AI) state. Methods: We have previously established AD and AI murine PC cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PC at phenotypic and subcellular levels. We statistically surveyed global protein expression profiles in these cell lines. Differential profiles were functionally interrogated by pathways and protein–protein interaction network analyses. Results: Protein expression pattern analysis revealed a total of 683 proteins, among which 99 were significantly differentially altered in PLum-AI cells as compared to PLum-AD cells (45 increased and 54 decreased). Principal component analysis (PCA) revealed that the two different cell lines clearly separated apart, indicating a significant proteome expression difference between them. Four of the proteins (vimentin, catalase, EpCAM, and caspase 3) that were differentially expressed in PLum-AI cells compared to PLum-AD cells were subjected to biochemical validation by Western blotting. Biological process gene ontology (GO) analysis of the differentially expressed proteins demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to PI3 kinase and androgen receptor pathways. Besides, other relevant biological processes that are enriched in PLum-AI cells included cell adhesion and cell migration processes, cell and DNA damage, apoptosis, and cell cycle regulation. Conclusions: Our protein expression analysis of a murine in vitro model of PC progression identified differential protein spots that denote this progression and that comprise high-potential targets for early treatment of PC with a personalized patient-specific approach. Efforts are underway to functionally assess the potential roles of these proteins as therapeutic targets for PC progression.

Down-regulation of PDGFRβ suppresses invasion and migration in osteosarcoma cells by influencing epithelial-mesenchymal transition.

Osteosarcoma (OS) is the most common malignant bone tumor primarily influencing children and adults. Approximately one-fifth of patients have micrometastasis in the lungs when OS is diagnosed. Platelet-derived growth factor receptor (PDGFR) beta (PDGFRβ) is a subtype of PDGFR. PDGFRβ has been noted to be highly expressed in OS cell lines and patient specimens, and is associated with metastasis and poor prognosis of OS. However, mechanistic insights into the exact role of PDGFRβ in OS pathogenesis and development are still lacking. Here we assessed the effects of PDGFRβ on invasive and migratory abilities, such as the epithelial-mesenchymal transition and phosphatidylinositol 3-kinase (PI3K), Akt and mammalian target of rapamycin (mTOR) pathways in HOS cells. Depleting PDGFRβ resulted in reduced migration of HOS cells in the small interfering RNA duplexes specific for the PDGFRβ group compared with the mock and scramble-treated groups in Transwell invasion assays. Using wound-healing assays, we demonstrate the rate of wound healing in the PDGF-BB-stimulated group was higher compared with the mock-treated group. Western blot showed that down-regulation of PDGFRβ decreased the expression of stromal phenotype markers and phosphorylation pathway proteins (PI3K, AKT and mTOR), but the epithelial phenotype marker was increased in HOS cells. Treating HOS cells with PDGF-BB revealed a treatment time-dependent increase of phosphorylated, but not total, PI3K, AKT and mTOR. Taken together, we suggest that PDGFRβ plays an important role in OS invasion, migration and epithelial-mesenchymal transition by influencing the PI3K, Akt and mTOR pathways, hence highlighting PDGFRβ as a potential therapeutic target for OS.

Inhibition of the PI 3-kinase pathway disrupts the unfolded protein response and reduces sensitivity to ER stress-dependent apoptosis.

Class Ia phosphoinositide 3-kinases (PI3K) are critical mediators of insulin and growth factor action. We have demonstrated that the p85α regulatory subunit of PI3K modulates the unfolded protein response (UPR) by interacting with and regulating the nuclear translocation of XBP-1s, a transcription factor essential for the UPR. We now show that PI3K activity is required for full activation of the UPR. Pharmacological inhibition of PI3K in cells blunts the ER stress-dependent phosphorylation of IRE1α and PERK, decreases induction of ATF4, CHOP, and XBP-1 and upregulates UPR target genes. Cells expressing a human p85α mutant (R649W) previously shown to inhibit PI3K, exhibit decreased activation of IRE1α and PERK and reduced induction of CHOP and ATF4. Pharmacological inhibition of PI3K, overexpression of a mutant of p85α that lacks the ability to interact with the p110α catalytic subunit (∆p85α) or expression of mutant p85α (R649W) in vivo, decreased UPR-dependent induction of ER stress response genes. Acute tunicamycin treatment of R649W+/- mice revealed reduced induction of UPR target genes in adipose tissue, whereas chronic tunicamycin exposure caused sustained increases in UPR target genes in adipose tissue. Finally, R649W+/- cells exhibited a dramatic resistance to ER stress-dependent apoptosis. These data suggest that PI3K pathway dysfunction causes ER stress that may drive the pathogenesis of several diseases including Type 2 diabetes and various cancers.

3050 – DELETION OF PHOSPHOINOSITIDE 3-KINASE DYSREGULATES AUTOPHAGY IN HEMATOPOIETIC STEM CELLS AND PROMOTES MYELOID DYSPLASIA

Adult hematopoietic stem cells (HSCs) maintain blood system through a delicate equilibrium between self-renewal and differentiation. Most hematopoietic growth factors and cytokines signal through the phosphoinositide 3-kinase (PI3K) pathway via three Class IA catalytic PI3K isoforms (P110α, β, and δ). However, it is not known if PI3K is required for HSC differentiation or self-renewal. We found that individual PI3K isoforms are dispensable in HSCs. To determine the redundant roles of PI3K isoforms in HSCs, we generated a triple knockout (TKO) mouse model with conditional deletion of P110α and P110β in hematopoietic cells, and germline deletion of P110δ. TKO mice develop transplantable pancytopenia, and TKO bone marrow cells have defective long-term multilineage repopulation. Surprisingly, we observed a significant expansion of donor TKO HSCs in transplant recipients. TKO bone marrow cells have decreased myeloid differentiation capacity and extended serial re-plating activity. Additionally, TKO bone marrow cells have dysplastic features similar to those seen in myelodysplastic syndrome (MDS) with multiple chromosomal abnormalities. For the maintenance of self-renewal of HSCs autophagy was shown to be essential. However, in TKO HSCs upon serum starvation autophagy is defective. Surprisingly, we found that in TKO cKit+ cells MTOR signaling is hyperactivated. Given that MTOR is a negative regulator of autophagy, this is consistent with the decrease in autophagy in TKO HSCs. The MTOR inhibitor rapamycin can induce autophagy in TKO HSCs, which rescues the self-renewal and differentiation defects caused by PI3K deletion. Our results uncover important roles of PI3K in regulating autophagy induction in HSCs to maintain the balance between self-renewal and differentiation. This work has potential implications for the treatment of MDS by regulating autophagy induction.

Progress in the management of endometrial cancer (subtypes, immunotherapy, alterations in PIK3CA pathway): data and perspectives.

Changes in molecular classification together with a deeper knowledge of both immune disregulation and phosphatidylinositol-3 kinase (PI3K) pathway alterations are leading to a new endometrial cancer treatment paradigm. This review will address the cutting-edge data in this field. This article will cover the updated data in endometrial cancer molecular classification and its correlation with the outcomes in randomized clinical trials (e.g., PORTEC-3). Moreover, we will review the latest data regarding checkpoint blockade molecules (CPB) in the recurrent setting and how they are changing the treatment landscape. In addition, the role of the PI3K inhibitors, their activity, and toxicity profile will be described. As result of the incorporation of molecular classification in our daily practice, the adjuvant treatment in endometrial cancer is rapidly evolving and leading to a new paradigm. The promising data observed with CPB in the recurrent setting have led to the food and drug administration approval of pembrolizumab as monotherapy and in combination with lenvatinib. Additionally, the current outcomes achieved with PI3K inhibitor agents encourage us to continue our clinical research to identify those patients who may benefit the most.

Increased PIP3 activity blocks nanoparticle mRNA delivery.

The biological pathways that affect drug delivery in vivo remain poorly understood. We hypothesized that altering cell metabolism with phosphatidylinositol (3,4,5)-triphosphate (PIP3), a bioactive lipid upstream of the metabolic pathway PI3K (phosphatidylinositol 3-kinase)/AKT/ mTOR (mammalian target of rapamycin) would transiently increase protein translated by nanoparticle-delivered messenger RNA (mRNA) since these pathways increase growth and proliferation. Instead, we found that PIP3 blocked delivery of clinically-relevant lipid nanoparticles (LNPs) across multiple cell types in vitro and in vivo. PIP3-driven reductions in LNP delivery were not caused by toxicity, cell uptake, or endosomal escape. Interestingly, RNA sequencing and metabolomics analyses suggested an increase in basal metabolic rate. Higher transcriptional activity and mitochondrial expansion led us to formulate two competing hypotheses that explain the reductions in LNP-mediated mRNA delivery. First, PIP3 induced consumption of limited cellular resources, "drowning out" exogenously-delivered mRNA. Second, PIP3 triggers a catabolic response that leads to protein degradation and decreased translation.

Class IA PI3K regulatory subunits: p110-independent roles and structures.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical regulator of many cellular processes including cell survival, growth, proliferation and motility. Not surprisingly therefore, the PI3K pathway is one of the most frequently mutated pathways in human cancers. In addition to their canonical role as part of the PI3K holoenzyme, the class IA PI3K regulatory subunits undertake critical functions independent of PI3K. The PI3K regulatory subunits exist in excess over the p110 catalytic subunits and therefore free in the cell. p110-independent p85 is unstable and exists in a monomer-dimer equilibrium. Two conformations of dimeric p85 have been reported that are mediated by N-terminal and C-terminal protein domain interactions, respectively. The role of p110-independent p85 is under investigation and it has been found to perform critical adaptor functions, sequestering or influencing compartmentalisation of key signalling proteins. Free p85 has roles in glucose homeostasis, cellular stress pathways, receptor trafficking and cell migration. As a regulator of fundamental pathways, the amount of p110-independent p85 in the cell is critical. Factors that influence the monomer-dimer equilibrium of p110-independent p85 offer additional control over this system, disruption to which likely results in disease. Here we review the current knowledge of the structure and functions of p110-independent class IA PI3K regulatory subunits.

The PI3K pathway induced by αMSH exerts a negative feedback on melanogenesis and contributes to the release of pigment.

The melanocortin-1 receptor (MC1R) belongs to the family of the G protein-coupled receptor (GPCR). Activated GPCRs can promote the phosphoinositide 3-kinase (PI3K) pathway. Few studies deal with the role of the PI3K pathway activation in response to αMSH. On B16-F10 cell line, we investigated the αMSH-dependent modulation of pAKT/AKT, as a key element of the PI3K pathway after rapid and prolonged stimulation. We demonstrated that αMSH triggers a rapid modulation of AKT which culminates in an increase in its phosphorylation. We highlighted a comparable upregulation of pAKT after exposure to αMSH on primary cultures of normal human melanocytes (NHMs) expressing a wild-type MC1R. On B16-F10 cells, NHMs, and an ex vivo model of human skin biopsies, we explored the influence of PI3K/AKT signaling triggered by αMSH, focusing on the control of melanogenesis and pigment release. We showed that the αMSH-dependent PI3K/AKT pathway exerts a negative feedback on melanogenesis and promotes the extracellular release of pigment. We strengthened the role of the PI3K/AKT pathway triggered by αMSH in preserving redox equilibrium and genome integrity, highlighting its role in affecting cell survival.

Combined targeting of G protein‐coupled receptor and EGF receptor signaling overcomes resistance to PI3K pathway inhibitors in PTEN‐null triple negative breast cancer

Triple‐negative breast cancer (TNBC) has poorer prognosis compared to other types of breast cancers due to the lack of effective therapies and markers for patient stratification. Loss of PTEN tumor suppressor gene expression is a frequent event in TNBC, resulting in over‐activation of the PI 3‐kinase (PI3K) pathway and sensitivity to its inhibition. However, PI3K pathway inhibitors show limited efficacy as monotherapies on these tumors. We report a whole‐genome screen to identify targets whose inhibition enhanced the effects of different PI3K pathway inhibitors on PTEN‐null TNBC. This identified a signaling network that relies on both the G protein‐coupled receptor for thrombin (PAR1/F2R) and downstream G protein βγ subunits and also epidermal growth factor receptor (EGFR) for the activation of the PI3K isoform p110β and AKT. Compensation mechanisms involving these two branches of the pathway could bypass PI3K blockade, but combination targeting of both EGFR and PI3Kβ suppressed ribosomal protein S6 phosphorylation and exerted anti‐tumor activity both in vitro and in vivo, suggesting a new potential therapeutic strategy for PTEN‐null TNBC.