PI3K Pathway Inhibitors Research Articles

\u03b22-AR blockade potentiates MEK1/2 inhibitor effect on HNSCC by regulating the Nrf2-mediated defense mechanism

The β2-Adrenergic receptor (β2-AR) is a G protein-coupled receptor (GPCR), involved in the development of many cancers, among which HNSCC. In this contest, β2-AR signaling interacts with different pathways, such as PI3K and MAPK, commonly activated by TK receptors. For this reason, TK blockade is one of the most adopted therapeutic strategies in HNSCC patients. In our study we investigated the effects of the β2-AR blocking in HNSCC cell lines, using the selective inhibitor ICI118,551 (ICI), in combination with the MAPK inhibitor U0126. We found that ICI leads to the blocking of p38 and NF-kB oncogenic pathways, strongly affecting also the ERK and PI3K pathways. Cotreatment with U0126 displays a synergic effect on cell viability and pathway alteration. Interestingly, we found that the β2-AR blockade affects Nrf2-Keap1 stability and its nuclear translocation leading to a drastic ROS increase and oxidative stress. Our results are confirmed by a TCGA dataset analysis, showing that NFE2L2 gene is commonly overexpressed in HNSC, and correlated with a lower survival rate. In our system, the PI3K pathway inhibition culminated in the blocking of pro-survival autophagy, a mechanism normally adopted by cancer cells to became less responsive to the therapies. The mTOR expression, commonly upregulated in HNSC, was reduced in patients with disease-recurrence. It is well known that mTOR has a strong autophagy inhibition effect, therefore its downregulation promoted pro-survival autophagy, with a related increase recurrence rate. Our findings highlight for the first time the key role of β2-AR and related pathway in HNSCC cell proliferation and drug resistance, proposing it as a valuable therapeutic molecular target.

Abstract 659: Synergistic antitumor activity of futibatinib (TAS-120), a FGFR1-4 inhibitor, and PI3K pathway inhibitors

Abstract Background: Futibatinib, a selective irreversible small-molecule fibroblast growth factor receptor (FGFR) 1–4 inhibitor is currently being investigated in phase 2 studies in patients with various FGFR-deregulated cancers. FGFRs use the PI3K pathway to promote cell growth and survival, suggesting that a combination of futibatinib with PI3K pathway inhibitors may result in synergistic activity. Therefore, we investigated the antitumor effects of futibatinib plus PI3K pathway inhibitors, everolimus (mTOR inhibitor) or MK2206 (AKT inhibitor). Methods: Human endometrial FGFR2-N549K-mutated AN3CA cancer cells were treated with futibatinib plus either everolimus or MK2206 or with each agent alone. AKT, ERK, mTOR, and S6 phosphorylation, cell viability, and apoptosis (via caspase-3/7 activity) were assessed. Cell growth inhibition was measured and combination indices (CIs) determined using CalcuSyn software; a CI<0.9 indicates synergy. Antitumor activity of futibatinib plus everolimus in ANC3A xenografts was evaluated by assessing relative tumor volume (RTV). Results: In ANC3A cells, the combination of futibatinib with MK2206 inhibited AKT phosphorylation; futibatinib plus either MK2206 or everolimus inhibited mTOR phosphorylation to a greater degree than monotherapy. Apoptosis induction by caspase-3/7 was readily detected with futibatinib plus MK2206, but low to no activity was observed with either agent alone. A synergistic effect on cell growth inhibition was observed for futibatinib plus everolimus (CI=0.26) or MK2206 (CI=0.14). In AN3CA xenografts, there was a significant (P<0.05) decrease in RTV with futibatinib plus everolimus vs monotherapy (Table). Table.Relative tumor volume in AN3CA mouse xenografts treated with futibatinib or everolimus or combination therapyTreatment (mg/kg/d)Relative tumor volumeDay 0Day 3Day 6Day 10Day 15Control14.657.8116.4937.77Futibatinib (15)a13.084.516.8412.59Futibatinib (50)a12.292.703.154.76Everolimus (2)a12.664.046.3410.24Futibatinib + Everolimus (15 + 2)b11.841.921.681.91Futibatinib + Everolimus (50 + 2)b11.501.310.840.72aDays 1–14 (oral);bDays 1–14 + Days 1–14 (both oral). Conclusions: The combination of futibatinib plus a PI3K pathway inhibitor resulted in synergistic antitumor effects. These data are encouraging and can serve as a basis for investigating the combination of PI3K pathway inhibitors with futibatinib. Citation Format: Akihiro Miura, Hiroshi Sootome, Toshiharu Komori, Hiroaki Ochiiwa, Hiroshi Hirai. Synergistic antitumor activity of futibatinib (TAS-120), a FGFR1-4 inhibitor, and PI3K pathway inhibitors [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 659.

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 < 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< 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 < 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.

Abstract 1090: Regulation of PTEN translation by PI3K signaling maintains pathway homeostasis

Abstract The PI3K pathway is a key regulator of metabolism, cell proliferation and migration and some of its components (e.g. PIK3CA and PTEN) are frequently altered in cancer by genetic events that deregulate its output. However, PI3K signaling is not usually the primary driver of these tumors and inhibitors of components of the pathway have only modest antitumor effects. We now show that both physiologic and oncogenic activation of the PI3K signaling by growth factors and an activating hotspot PIK3CA mutation respectively, cause an increase in the expression of the lipid phosphatase PTEN, thus limiting the duration of the signal and the output of the pathway in tumors. Pharmacologic and physiologic inhibition of the pathway by HER2/PI3K/AKT/mTOR inhibitors and nutrient starvation respectively reduce PTEN, thus buffering the effects of inhibition and contributing to the rebound in pathway activity that occurs in tumors. This regulation is found to be a feature of multiple types of cancer, non-cancer cell line and PDX models thereby highlighting its role as a key conserved feedback loop within the PI3K signaling network, both in vitro and in vivo. Regulation of expression is due to mTOR/4EBP1 dependent control of PTEN translation and is lost when 4EBP1 is knocked out. Translational regulation of PTEN is therefore a major homeostatic regulator of physiologic PI3K signaling and plays a role in reducing the output of oncogenic mutants that deregulate the pathway and the antitumor activity of PI3K pathway inhibitors. Citation Format: Radha Mukherjee, Kiran Gireesan Vanaja, Jacob A. Boyer, Juan Qiu, Xiaoping Chen, Elisa de Stanchina, Sarat Chandarlapaty, Andre Levchenko, Neal Rosen. Regulation of PTEN translation by PI3K signaling maintains pathway homeostasis [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 1090.

Abstract NG16: Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kα inhibitors

Abstract NG16: Double <i>PIK3CA</i> mutations in <i>cis</i> increase oncogenicity and sensitivity to PI3Kα inhibitors

8-prenylgenistein exerts osteogenic effects via ER α and Wnt-dependent signaling pathway

8-prenylgenistein (8PG) was previously reported to exert stronger osteogenic activity than genistein, a well-known soy phytoestrogen. However, the molecular mechanism underlying the actions of 8PG on osteoblasts was far from clear. In the present study, the osteogenic effects and mechanisms of 8PG and genistein were studied using human BMSC and murine pre-osteoblast MC3T3-E1 cells. Our results indicated that the stimulatory effects of 8PG and genistein on osteoblast differentiation were abolished by co-incubation with MPP (10−6 M, an ERα antagonist), but not PHTPP (10−6 M, an ERβ antagonist). Molecular docking indicated that the binding mode of 8PG toward ERα was similar to that of genistein and therefore could not account for their differential osteogenic actions. In silico target profiling identified the involvement of glycogen synthase kinase-3β (GSK-3β), a key mediator of Wnt/β-catenin pathway, in the actions of 8PG. However, instead of directly inhibiting GSK-3β enzymatic activities, 8PG and genistein were found to induce GSK-3β phosphorylation at Serine-9 in osteoblastic MC3T3-E1 cells. 8PG exerted more potent effects than genistein in stimulating expressions of LRP5, β-catenin, Runx2, osteocalcin, alp, opg, major protein and gene markers involved in Wnt signaling pathway in MC3T3-E1 cells. Moreover, the inhibition of Wnt signaling by DKK1 could be restored by treatment with 8PG and genistein. However, 8PG, but not genistein, stimulated ERα-dependent β-catenin protein expression in MC3T3-E1 cells. Furthermore, the increase in ALP activity, LRP5 and phospho-Akt/Akt expression by 8PG and genistein were abolished by co-treatment with LY294002 (10−5 M, a PI3K pathway inhibitor). Collectively, our results suggested that the osteogenic activities of 8PG was mediated by GSK-3β phosphorylation through the induction of Wnt/β-catenin and ERα-associated PI3K/Akt signaling.

BRCA1 subcellular localization regulated by PI3K signaling pathway in triple-negative breast cancer MDA-MB-231 cells and hormone-sensitive T47D cells.

This study is to investigate the effect of the PI3K/Akt signaling pathway on the regulation of BRCA1 subcellular localization in triple-negative breast cancer (TNBC) MDA-MB-231 cells and hormone-sensitive T47D cells. We found that heregulin-activated T47D cells showed more nuclear localization of BRCA1, but BRCA1 nuclear localization decreased after the inhibition of the PI3K signaling pathway. In MDA-MB-231 cells, activation or inhibition of the PI3K signaling pathway did not significantly affect cell apoptosis and BRCA1 nuclear translocation (P > 0.05). However, in T47D cells, the activation of the PI3K pathway significantly increased cell apoptosis (P < 0.05). In the heregulin-activated MDA-MB-231 and T47D cells, the phosphorylation of Akt and BRCA1 was significantly increased (P < 0.05), while that was significantly reduced after PI3K pathway inhibition (P < 0.05). The changing trends of the mRNA levels of Akt and BRCA1 in MDA-MB-231 and T47D cells after PI3K pathway activation or inhibition were consistent with the trends of their proteins. In both MDA-MB-231 and T47D cells, BRCA1 phosphorylation is regulated by the PI3K signaling pathway, but the nuclear localization of BRCA1 is different in these two cell lines. Moreover, the apoptosis rates of these two cell lines are different.

A Genome-scale CRISPR Screen Identifies the ERBB and mTOR Signaling Networks as Key Determinants of Response to PI3K Inhibition in Pancreatic Cancer.

KRAS mutation is a key driver of pancreatic cancer and PI3K pathway activity is an additional requirement for Kras-induced tumorigenesis. Clinical trials of PI3K pathway inhibitors in pancreatic cancer have shown limited responses. Understanding the molecular basis for this lack of efficacy may direct future treatment strategies with emerging PI3K inhibitors. We sought new therapeutic approaches that synergize with PI3K inhibitors through pooled CRISPR modifier genetic screening and a drug combination screen. ERBB family receptor tyrosine kinase signaling and mTOR signaling were key modifiers of sensitivity to alpelisib and pictilisib. Inhibition of the ERBB family or mTOR was synergistic with PI3K inhibition in spheroid, stromal cocultures. Near-complete loss of ribosomal S6 phosphorylation was associated with synergy. Genetic alterations in the ERBB-PI3K signaling axis were associated with decreased survival of patients with pancreatic cancer. Suppression of the PI3K/mTOR axis is potentiated by dual PI3K and ERBB family or mTOR inhibition. Surprisingly, despite the presence of oncogenic KRAS, thought to bestow independence from receptor tyrosine kinase signaling, inhibition of the ERBB family blocks downstream pathway activation and synergizes with PI3K inhibitors. Further exploration of these therapeutic combinations is warranted for the treatment of pancreatic cancer.

Oncogene-independent BCR-like signaling adaptation confers drug resistance in Ph-like ALL.

Children and adults with Philadelphia chromosome-like B cell acute lymphoblastic leukemia (Ph-like B-ALL) experience high relapse rates despite best-available conventional chemotherapy. Ph-like ALL is driven by genetic alterations that activate constitutive cytokine receptor and kinase signaling, and early-phase trials are investigating the potential of the addition of tyrosine kinase inhibitors (TKIs) to chemotherapy to improve clinical outcomes. However, preclinical studies have shown that JAK or PI3K pathway inhibition is insufficient to eradicate the most common cytokine receptor-like factor 2-rearranged (CRLF2-rearranged) Ph-like ALL subset. We thus sought to define additional essential signaling pathways required in Ph-like leukemogenesis for improved therapeutic targeting. Herein, we describe an adaptive signaling plasticity of CRLF2-rearranged Ph-like ALL following selective TKI pressure, which occurs in the absence of genetic mutations. Interestingly, we observed that Ph-like ALL cells have activated SRC, ERK, and PI3K signaling consistent with activated B cell receptor (BCR) signaling, although they do not express cell surface μ-heavy chain (μHC). Combinatorial targeting of JAK/STAT, PI3K, and "BCR-like" signaling with multiple TKIs and/or dexamethasone prevented this signaling plasticity and induced complete cell death, demonstrating a more optimal and clinically pragmatic therapeutic strategy for CRLF2-rearranged Ph-like ALL.

Antitumor activity of tipifarnib and PI3K pathway inhibitors in HRAS-associated HNSCC.

e15658 Background: HRAS is an important driver oncogene in HNSCC and other squamous cell carcinomas (SCCs). HRAS mutations are observed in 6-10% of HNSCC cases and appear to define a unique subset of HNSCC associated with wild-type TP53, co-mutations in caspase 8 and a characteristic methylation pattern. RAS proteins undergo several posttranslational modifications that facilitate their attachment to membranes. The first and most critical modification is the addition of a farnesyl isoprenoid moiety by the enzyme farnesyltransferase (FT), prompting the development of FTIs for the treatment of RAS-driven cancers. Tipifarnib is a potent and selective FTI that has demonstrated compelling antitumor activity in a heavily pretreated cohort of recurrent/metastatic HNSCC carrying HRAS mutations. Based on these data, a pivotal study (NCT03719690) evaluating the efficacy of tipifarnib in HRAS mutant HNSCC (AIM-HN) is currently ongoing. Methods: HRAS is the predominant RAS in squamous epithelial cells and SCCs commonly overexpress the protein. In TCGA PanCancer Atlas, approximately 30% of HNSCC cases have elevated HRAS gene expression, raising the possibility that some HRAS wild-type (WT) HNSCCs may also display a degree of dependence on this oncoprotein. The PI3K-AKT-mTOR pathway is another prominent driver in HNSCC, and reports suggest HRAS and PI3K can each drive resistance to inhibitors of the alternative pathway. In this study we explored the activity of tipifarnib in cellular models and six HRAS-mutant and twelve HRAS WT HNSCC patient-derived xenograft (PDX) models alone and in combination with inhibitors of PI3K-a (BYL-719), AKT (GSK2141795) and mTORC1/2 (INK128). Results: Tipifarnib induced regression in several HRAS WT-overexpressing models. In those cases where the FTI was only moderately active, robust inhibition of tumor growth was observed in the majority of animals treated with the combination of tipifarnib and the PI3K-a inhibitor BYL-719. Similar activity was noted in a smaller panel with GSK2141795 &amp; INK128. Regressions with the FTI-PI3K-a inhibitor doublet were observed both in tumors that were WT or harbored hotspot mutations or amplification of the PIK3CA gene and in HRAS mutants that carried or lacked PIK3CA mutations. Conclusions: Concomitant blockade of both targets may have surprisingly broad and potent anti-tumor activity in HNSCC. Additional biomarkers associated with sensitivity or resistance and alterations in gene expression (by RNAseq) and oncogenic signaling following treatment either as a monotherapy or in combinations will also be described.

Temsirolimus combined with etoposide and cyclophosphamide for relapsed/refractory acute lymphoblastic leukemia: Therapeutic advances in Childhood Leukemia Consortium (TACL 2014-001) trial.

10512 Background: PI3K/mTOR signaling, a critical pathway in cell proliferation, metabolism, and apoptosis, is often dysregulated in acute lymphoblastic leukemia (ALL). A phase 1 trial of the mTOR inhibitor temsirolimus combined with etoposide and cyclophosphamide was performed in children with relapsed/refractory (r/r) ALL. Methods: Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily days 1-5. The starting dose level (DL) of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). MRD was performed centrally. PI3K pathway inhibition was measured by phosphoflow cytometry (PFC) analysis of peripheral blood (PB) from treated patients (pts). Results: Sixteen heavily pretreated r/r ALL pts ages 2-19 years with marrow blasts &gt; 25% were enrolled; 15 were evaluable [10 B-ALL/5 T-ALL]. One dose-limiting toxicity (DLT) of grade (Gr) 4 pleural and pericardial effusions with pneumonitis/lung infection leading to Gr 5 cardiorespiratory arrest occurred in a pt treated at DL3. No further DLTs were seen in the DL3 expansion and DL4 cohorts. Gr 3/4 non-hematologic toxicities occurring in ≥ 3 pts included febrile neutropenia, elevated ALT, hypokalemia, mucositis, and tumor lysis syndrome and were independent of dose. Of 15 evaluable pts, 4 (27%; 2 B-ALL/2 T-ALL) had a complete response (CR) after cycle 1, comprised of 1 pt at each DL. Three had MRD &lt; 0.01%. Three pts (20%; 2 B-ALL/1 T-ALL) had partial response (PR). Overall response rate (CR+PR = ORR) was 47%. Pharmacodynamic PFC studies compared phosphoprotein levels pre (day 0) and post treatment (days 3-5) in 9 consenting pts with available PB. All tested pts showed basal activation of PI3K pathway signaling. Dose-dependent inhibition of mTOR targets phosphorylated (p) S6 and/or p4EBP1 was observed in 9/9 and 6/9 pts, respectively, following temsirolimus and chemotherapy treatment. Various patterns of compensatory upregulation of pPI3K, pmTOR, pAkt, and/or pERK was observed. Conclusions: Temsirolimus at 25 mg/m2 combined with salvage etoposide and cyclophosphamide has an acceptable safety profile in high-risk pediatric patients with r/r ALL. Responses were observed at all DLs. mTOR target inhibition was achieved and appeared to correlate with dose level. Future testing of other PI3K/mTOR pathway inhibitors in combination with chemotherapy may be warranted with a goal of further increasing response in r/r ALL. Clinical trial information: NCT01614197.

Phase II study of copanlisib in patients with tumors with PIK3CA mutations (PTEN loss allowed): NCI MATCH EAY131-Z1F.

3506 Background: The NCI-MATCH (EAY131) is a platform trial that enrolls patients (pts) with solid tumors, lymphomas, or multiple myeloma to targeted therapies based on matching genomic alterations of interest (NCT02465060). Arm Z1F evaluated copanlisib, a highly selective, pan-Class 1 PI3K inhibitor with predominant activity against both the δ and α isoforms in pts with PIK3CA mutations. Methods: Pts received copanlisib (60 mg IV) on days 1, 8, and 15 in 28-day cycles until progression/toxicity. Tumor assessment was every 2 cycles. The primary endpoint was objective response rate (ORR); secondary endpoints were PFS, 6-month PFS, and predictive biomarkers. Pts with KRAS mutations, HER2+ve breast cancers, lymphomas were excluded. Results: 35 pts were enrolled (from 8/2/18 to 12/27/18), of which, 28 pts were available for analysis (7 patients, not eligible or did not start therapy). Multiple histologies were enrolled with gynecologic (n = 7), gastrointestinal (n = 6), and genitourinary (n = 5) the most common tumors. Median age 61 (range 42-78). 75% of pts had ≥ 3 lines of prior therapy. 54% of PIK3CA mutations were located in the helical domain, 32% in kinase domain and 14% in other domains. Twenty-six pts had co-occurring gene alterations (median 3; range 1-9), with 9 patients having 4 or more gene alterations. The ORR was 11% (3/28, 90% CI: 3%-25%). Partial responses were seen in uterine cancer, clear cell carcinoma of anterior abdominal wall, and liposarcoma. 6 pts had &gt; 6 months of stable disease and clinical benefit rate was 32% (9/28). Two pts are still on treatment. The most common reason for protocol discontinuation was disease progression (n = 18, 69%). Thirty pts were included for toxicity analysis. Ten pts (33%) had grade 1 or 2 toxicities, 16 pts (53%) had grade 3 toxicities, and one patient (3%) had grade 4 toxicity (CTCAE v5.0). Most common toxicities include hyperglycemia (n = 19), fatigue (n = 11), hypertension (n = 10), diarrhea (n = 10), and nausea (n = 9). Total of 5 deaths were reported, none related to treatment. Conclusions: Copanlisib showed meaningful clinical activity across various tumors with PIK3CA mutation in the late-line refractory setting. Further study either alone or in combinations in select tumors is warranted. G3/4 toxicities observed were consistent with reported toxicities for PI3K pathway inhibition. Clinical trial information: NCT02465060 .

Tetramethylpyrazine ameliorates hepatic fibrosis through autophagy-mediated inflammation.

Imbalanced immune response and hepatic fibrosis are key factors related to the progression of chronic liver diseases. Tetramethylpyrazine (TMP), a natural alkaloid, has been widely used for treating liver injury. In this study, we explored the effect of TMP on hepatic fibrosis and the related mechanisms regulating autophagy. A rat model of hepatic fibrosis and a model using an hepatic stellate cell line (HSC-T6) were created using CCl4 and platelet-derived growth factor (PDGF). Staining with haematoxylin and eosin (HE), Masson's stain, and TUNEL were performed for pathological diagnosis. ELISA, Western blotting, and immunofluorescence analyses were conducted to determine the expression levels of the specific markers for fibrosis, autophagy, inflammation, and signalling pathways. TMP treatment significantly rescued pathological injury and hepatic fibrosis. It also alleviated imbalances in the immune system, accumulation of extracellular matrix, and autophagy signals in hepatic fibrosis. At the same time, we found that application of the autophagy inducer rapamycin enhanced the therapeutic effect of TMP, whereas the autophagy inhibitor 3-methyladenine, PI3K pathway inhibitor LY294002, and AKT pathway agonist SC79 did the opposite. TMP exerts therapeutic effects in hepatic fibrosis mainly through promoting autophagy to ameliorate inflammation by inhibiting the AKT-mTOR signalling pathway, providing a new perspective for the treatment of chronic liver diseases.

ALDH1A3 serves as a predictor for castration resistance in prostate cancer patients

BackgroundAldehyde dehydrogenase 1A3 (ALDH1A3) has been implicated in the survival and proliferation of prostate cancer cells.MethodsWe retrospectively reviewed our patients with advanced disease on adjuvant hormonal therapy after prostatectomy. Time to castration resistance stage was documented. And Immunohistochemistry analysis for ALDH1A3 was performed for those patient samples on tissue microarray. Bioinformatics anslysis was used for RNA sequencing data of both primary prostate cancer and metastatic castration resistance prostate cancer (mCRPC) from online datasets. Crispr-Cas9 was used to knock out ALDH1A3 in prostate cancer luminal cells, and morphologic analysis as well as the Gene Set Enrichment Analysis (GSEA) were facilitated to discover the mechanisms of the resistance phenotype.ResultsWe found that the patients with ALDH1A3 low expression had shorter time to progression to castration resistance compared with those of higher expression group on adjuvant hormonal therapy after radical prostatectomy. The ALDH1A3 knockout cells gradually acquired resistance to androgen deprivation therapy, a few cells have been found in knockout group showing as that the spindle-like luminal cells in charcoal stripped medium. Furthermore, PI3K pathway activation has been confirmed by Western blot. The PI3K pathway inhibitor BEZ235 has been demonstrated that the acquired ADT resistance by ALDH1A3 down regulation could be rescued by PI3K pathway inhibitor.ConclusionThese results suggested a novel function for ALDH1A3 in development of mCRPC, and indicated PI3K pathway inhibitor has the potential in the treatment of a subgroup of mCRPC patients.

Abstract B31: Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer

Abstract With KRAS mutations found in 95% of pancreatic ductal adenocarcinoma (PDAC), an effective anti-KRAS therapeutic strategy is anticipated to make a significant impact on the treatment of PDAC. Among the major directions currently being pursued, inhibitors of KRAS effector signaling are believed to be the most promising. However, as monotherapy, effector inhibitors have not demonstrated significant activity and concerns with normal tissue toxicity remain. Combination inhibitor strategies are considered the best approach to overcome these limitations. To identify effector inhibitor-based combinations, we applied a 525-oncology drug screen to identify combinations that enhanced the cytotoxic activity of inhibitors of the RAF-MEK-ERK and the PI3K-AKT-mTOR effector pathways. While many cytotoxic combinations were identified with RAF effector pathway inhibitors, few were identified with PI3K pathway inhibitors. The same classes of inhibitors were identified with RAF, MEK and ERK inhibitors. In addition to inhibitors of the PI3K-AKT-mTOR pathway, microtubule, HDAC and HSP90 inhibitors synergistically enhanced RAF and ERK inhibitor antitumor activity. Addressing a basis for synergy, we found that the different combinations showed enhanced loss of MYC protein, a key ERK substrate. In summary, our studies identify promising effector inhibitor-based combinations for PDAC treatment. Citation Format: Irem Ozkan-Dagliyan, Craig M. Goodwin, Kirsten L. Bryant, Samuel D. George, Kelly Lucas, Prson Gautam, Krister Wennerberg, Adrienne D. Cox, Channing J. Der. Combination inhibitor strategies targeting KRAS effector signaling in KRAS-mutant pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B31.

Altered Gut DNA virome diversity associated HMGB1 release regulates reactive Astrocytes-induced IL6 release preferably via TLR4-NFkB pathway in experimental Gulf War Illness

Abstract In a recent study, we showed that the gut DNA bacteriophage dysbiosis in Gulf War Illness (GWI) was strongly associated with compromised intestinal epithelial cell integrity, increased circulatory IL6 and neuroinflammation. The current study further investigates the mechanism of DNA bacteriophage-IL6 axis in neuroinflammation. Advancing the previous findings, we show that viral dysbiosis positively correlated with high mobility group box protein 1 (HMGB1; a damage-associated molecular pattern) expression and release in circulation following GWI induction in mice. The circulatory HMGB1 activated brain Astrocytes via altering brain endothelial tight junction proteins and crossing the blood-brain barrier. Interestingly, both in GWI mice and mouse primary Astrocytes cell culture showed an increased brain IL6 mRNA and subsequent protein expression. However, GWI mice treated with Ribavirin (used for partial gut viral sterility) showed decreased intestinal HMGB1 and brain IL6 expression. Mechanistically, HMGB1 activated innate immune response via IRAKs-IKKα-NFkB instead of either RAGE-MAPK or PI3K-mTOR pathway. Surprisingly, inhibition of RAGE or PI3K pathway in HMGB1 primed mouse Astrocyte cells showed a significant increase in IL6 expression and release suggesting NFkB activation as a preferential pathway in GWI-Astrocyte-induced IL6 release. In summary, GWI-associated gut viral dysbiosis associated intestinal HMGB1 release activates brain Astrocytes and IL6 release via toll-like receptor 4-NFkB dependent pathway, thus causing neuroinflammation in GWI. The above mechanism can form a basis for studying inflammation-associated neurocognitive abnormalities in GWI.

PI3K-dependent Reprogramming of Hexokinase Isoforms Regulates B Lymphocyte Metabolism

Abstract The PI3K signalling pathway is known to regulate B cell metabolic programming upon activation, but the mechanisms involved are not well understood. Here we find that the PI3K pathway controls reprogramming of hexokinases (HKs), the enzymes that convert glucose into glucose-6-phosphate as a key rate-limiting step of glycolysis and other metabolic pathways. In primary mouse B cells, PI3K pathway inhibition substantially impaired the activation-induced increase in extracellular acidification rates (ECAR), a measure of glycolysis. In contrast, B cells isolated from PI3Kdelta gain-of-function mutant mice exhibit elevated ECAR. We find that B cell activation substantially elevates protein levels of HK2 and HK3 isoforms, but not HK1, in a PI3K-dependant manner. PI3K or mTOR inhibition significantly reduced induction of HK2 and HK3 expression, whereas Akt inhibition did not affect HK isoform expression. In human B lymphoma cells, HK isoforms differ significantly in their degree of mitochondrial localization, with HK1 being mitochondrial, HK3 being cytoplasmic and HK2 present in both mitochondria and cytoplasm. To assess whether HK isoforms have unique non-redundant functions, HK2-deficient B lymphoma cells were generated and were found to exhibit decreased ECAR as well as significant changes in metabolomic profile, despite normal expression and localization of HK1 and HK3. Taken together, our study reveals that PI3K-dependant reprogramming of hexokinase isoforms can impact on B cell glycolysis and other metabolic pathways. Studies in progress are examining the functional importance of HK2 in antibody responses using mice with B cell-specific deletion of this enzyme.

Abstract OT2-06-01: A phase-2 trial of neoadjuvant alpelisib and nab-paclitaxel in anthracycline refractory triple negative breast cancers with PIK3CA or PTEN alterations

Abstract Background:While TNBC patients with pathologic complete response (pCR) or residual cancer burden (RCB-I) have good survival rates, those with extensive residual disease (RCB-II or RCB-III) after neoadjuvant chemotherapy (NACT) have poor prognosis. Patients without response to their first chemotherapeutic regimen have very low chance (5%) of achieving pCR. We have created a biomarker-driven drug development strategy, ARTEMIS (A Randomized, TNBC Enrolling trial to confirm Molecular profiling Improves Survival) to identify novel targeted therapies focusing on chemo-insensitive disease. The PI3K/Akt/mTOR pathway is a critical regulator of cell growth, apoptosis, metabolism in cancer cells and its activation is known to play an important role in resistance to chemotherapy. Clinical studies have shown that the combination of PI3K pathway inhibitors with taxanes are associated with improvement in response rates and survival. Alpelisib is approved for hormone receptor positive, HER2 negative, metastatic breast cancer patients with activating PIK3CA mutations. This study hypothesizes combination of alpelisib with nab-paclitaxel will improve PCR/RCB-1 in treatment-resistant early stage TNBC patients with activating PIK3CA or PTEN loss of function alterations. Trial Design: This will be a non-randomized, two- arm, open label, phase II study to evaluate the clinical activity of alpelisib and nab-paclitaxel in high-risk TNBC patients with PIK3CA or PTEN alterations with refractory or suboptimal response to anthracycline-based NACT. Eligibility criteria Stage 1-3 TNBC defined as ER&amp;lt;10%; PR&amp;lt;10% and HER2 negativeActivating alterations in PIK3CA, or PTEN lossResidual primary tumor size of at least 1.0 cm or evidence of lymph node involvement by imaging (ultrasound or MRI) after anthracycline-based NACTECOG performance status ≤1Normal organ and marrow functionReceived at least one dose of an anthracycline-based NACT (eligible if therapy was discontinued due to disease progression or therapy intolerance) Specific aims To determine if alpelisib in combination with nab-paclitaxel will improve rates of pathologic response (pCR/RCB-0 or RCB-I) from 5% to 20% in patients with chemotherapy insensitive TNBC with activating PIK3CA alterations (Arm-1) or PTEN loss of function alterations (Arm-2)To determine the radiographic response rate for alpelisib in combination with nab-paclitaxel in chemotherapy insensitive TNBC with PIK3CA (Arm-1) or PTEN (Arm-2) alterationsDetermine toxicity of alpelisib in combination with nab-paclitaxel given in the neoadjuvant settingDetermine progression free survival (PFS) at 3 years for patients treated with alpelisib in combination with nab-paclitaxel given in the neoadjuvant setting Statistical methods Primary objective is to determine if alpelisib in combination with nab-paclitaxel in the neoadjuvant setting will improve rates of pathologic response (counting pCR/RCB-0 or RCB-I as response) from 5% (control) to 20% (experimental arms). A Simon two-stage design will be employed with 12 patients in the first stage. If at least one patient has pCR/RCB-1, 25 more patients will be added for a total of 37 patients (alpha = beta = 10%). This design has a 54% probability of early termination after the first stage if the true pCR or RCB-I probability is 5%. Contact information for people with a specific interest in the trial sdamodaran@mdanderson.org Citation Format: Senthil Damodaran, Jennifer K Litton, Kenneth R. Hess, Colleen T. Eppig, Krzysztof J. Grzegorzewski, Funda Meric-Bernstam, Ignacio I. Wistuba, Jason B White, Gaiane M. Rauch, Rosalind P. Candelaria, Beatriz E. Adrada, Bora Lim, Stacy L. Moulder, Debu Tripathy. A phase-2 trial of neoadjuvant alpelisib and nab-paclitaxel in anthracycline refractory triple negative breast cancers with PIK3CA or PTEN alterations [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-06-01.

Abstract P4-08-05: Novel tumor suppressor regulating the PI3K/AKT pathway in triple negative breast cancer (TNBC)

Abstract The PI3K/AKT signaling pathway is a known oncogenic pathway in breast cancer, involved in essential cellular processes necessary for cancer development and progression, including altered proliferation, metabolism, and cell survival. Mutations in this pathway occur in 20% of all TNBC cases, making it the most deregulated pathway in the only subclass of breast cancer with no targeted therapy. Few clinical trials testing PI3K/AKT pathway inhibition in these breast cancers have shown limited long term efficacy; the heterogeneity of tumors and variable response rates in clinical studies underscore the necessity to better understand the mechanisms governing TNBC and the PI3K/AKT pathway in order to predict patient response to targeted therapies. In particular, appropriate biomarkers are required to identify the tumor subtypes that may be more responsive to PI3K pathway inhibition. While regulation of AKT activation through a classic phosphorylation signaling cascade is well understood, recent studies have revealed a non-canonical pathway contributing to regulate AKT activation and possibly providing an unexpected mechanism for resistance to PI3K inhibition. Non-canonical regulation of AKT is provided by non-proteolytic ubiquitination of AKT favoring its recruitment to the plasma membrane and leading to hyperactivation, but a full understanding of the mechanism and players involved in this process is missing. Our lab has recently identified G-Protein Pathway Suppressor 2 (GPS2) as a novel regulator of the insulin signaling pathway through the inhibition of AKT ubiquitination in the adipose tissue. Here, we explore the role of GPS2 as a tumor suppressor in the context of breast cancer. Deletion of GPS2 in aggressive MDA-MB231 TNBC cells results in increased AKT phosphorylation associated with greater proliferation, migration and invasion in vitro, with all phenotypes mitigated in presence of the allosteric AKT inhibitor MK2206.Transcriptomic profiling of wild-type MB231, MB231-GPS2-KO, and myristoylated AKT (MB231-myrAKT) revealed a significant overlap between the MB231-GPS2-KO and myrAKT signatures. Conversely, deletion of GPS2 in MDA-MB468, a TNBC cell line with an activating mutation in the PI3K/AKT pathway, do not show any increase in proliferation, migration or invasion, but remain sensitive to AKT inhibition. Together these results indicate that loss of GPS2-mediated inhibition of AKT ubiquitination is sufficient to drive activation of the PI3K/AKT pathway in absence of other activating mutations. Furthermore, preliminary analysis of an orthotopic model of MB231-GPS2-KO cells in NOD scid gamma (NSG) mice resulted in greater breast tumor sizes when compared to the parental cell line, confirming GPS2 putative tumor suppressor role in vivo. Further studies are required to investigate GPS2 expression levels and mutational status in tumor samples, and to confirm GPS2 relevance as a predictive biomarker for treatment outcome, thus exposing an additional category of tumors that could be responsive to PI3K/AKT class inhibition and/or would benefit from alternative strategies to prevent resistance. Citation Format: Stefanie Chan, Emma Smith, Stefano Monti, M Dafne Cardamone, Valentina Perissi. Novel tumor suppressor regulating the PI3K/AKT pathway in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-05.

Abstract P3-03-13: Testing algorithmic basis for combinations of targeted drugs by a mechanism-based laboratory friendly identification of apoptosis in live breast cancer cells

Abstract Background: The mechanism of induction of apoptosis has been one of the features to characterize and classify anti-tumor drugs. We have standardized a mechanism-based approach to identify apoptosis in live tumor cells by triple co-fluorescence (De et al., 2018; SCIENTIFIC REPORTS). Our protocol tests three crucial features of apoptosis; the enzymatic activity of executioner caspase3, cell surface presentation of caspase-dependent phosphatidylserine and mitochondrial function. Navigating the genomic landscape of tumors in patients with Triple Negative Breast Cancers (TNBC) at Avera Cancer Institute, we have identified algorithm for the rational combination of PI3K-pathway (PP) drugs. Aim: Here we present the results of testing the rational combinations of PP-targeted drugs on apoptosis using our mechanism-based laboratory friendly procedure. Methods: Our protocol co-stains live tumor cells in real time with NucView488 Casp3substrate (N), CF594AnnexinV (A), and MitoViewBlue (M). We have used carboplatin and targeted-drugs like olaparib, ABT888, BMN673, AZD2014, GDC0980, and AZD5363 in BRCA-WT (MDA-MB231, MDA-MB468) and BRCA-incompetent (SUM149) TNBC cells. Imaging of cells using simultaneous live/dead cell markers (Calcein AM green/EthD-1 red) was used for validation. Results: We retrospectively analyzed genomic data [FoundationOne] from 100 consecutive patients seen in our center from 2014. 137 genes were altered including 12 PP and 11 DNA-repair genes. We used AnnexinV-PE staining (Accuri C6), confocal IF imaging and WB expression of apoptotic signals and mitochondrial potential (TMRE-A) as validation criteria following different combinations. Treatment with the rational combination(s) like PARP inhibitor(s) plus carboplatin and PP inhibitor decreased proliferation, induced profound early apoptotic events, along with increased apoptotic signaling and increased mitochondrial depolarization in real time. Dual treatment also blocked cytoplasmic M staining while increasing both nuclear N substrate staining and membranous A staining. Control cells had higher Calcein AM positive (live) cells and fewer EthdD-1 stained red nuclei of dead cells than treated cells. Merged images (M+N+A) showed 100% mutual exclusivity between M and N or A stains in both control and treated cells as determined by overlap and colocalization coefficients. Conclusion: Our method of staining identified the apoptosis following treatment of drug-combination(s), which was based on the genomic alteration(s) present in TNBC patients. Citation Format: Nandini Dey, Jennifer Carlson Aske, Pradip De. Testing algorithmic basis for combinations of targeted drugs by a mechanism-based laboratory friendly identification of apoptosis in live breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-13.