PI3K Gamma Research Articles

New 1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-2-phenylisoquinoline-1(2H)-ones as Phosphoinositide 3-kinase Inhibitors for Treating Cancer and Other Diseases.

The patent describes novel useful compounds, such as PI3K protein kinase inhibitors, in particular as PI3K delta (δ) and/or gamma (γ) protein kinase modulators. The present disclosure also provides methods for preparing PI3K protein kinase inhibitors, pharmaceutical compositions containing them, and methods of treatment, prevention, and amelioration of PI3K kinase-mediated diseases, and disorders.

Dual Inhibition of PI3K Delta and PI3K Gamma Isoforms to Prevent Graft-Versus-Host Disease By Limiting T Cell Expansion In Vivo

Dual Inhibition of PI3K Delta and PI3K Gamma Isoforms to Prevent Graft-Versus-Host Disease By Limiting T Cell Expansion In Vivo

Immunosuppressive Mechanisms of Tumor-Associated Macrophages: Bioinformatic Analyses and Targeting

In recent years, the treatment of various cancers with immunotherapeutic strategies has revolutionized the classical treatments with chemotherapy or radiation. Such immunotherapeutic strategies are effective only in a subset of cancer patients who were unresponsive to conventional therapies and are not generalized to all cancer types. Several mechanisms have been reported that underlie the failure of the natural anti-tumor immunity or the administered immunotherapeutic agents in the treatment of cancer. Among these mechanisms is the pivotal role played by the immunosuppressive tumor microenvironment (TME). The TME is complex and consists of the stroma, blood vessels, and several cell types that have a direct relationship with the tumor as well as the tumor-relationship with the TME. Among the immunosuppressive cells in the TME are the tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs), and cancer-associated fibroblasts (CAFs). These cells altogether inhibit both innate and the adaptive anti-tumor immune responses. Noteworthy, TAMs represent > 50 % of all the infiltrating cells in the TME and their frequencies correlate with poor prognoses in many cancers. The depletion or inactivation of TAMs has been reported to restore, in large part, the anti-tumor immune response in several cancers. In this review, we discuss (i) the interrelationship between TAMs and cancer stem cells, (ii) the various mechanisms by which TAMs suppress the immune response [e.g., expression of inhibitory receptors and ligands, secretion of immunosuppressive cytokines, secretion of chemokines, secretion of arginase 1, secretion of IDO1 and expression of the triggering receptor expressed on myeloid cells (TREM)], and (iii) targeting TAMs for immunotherapy (e.g., depletion of TAMs, killing of TAMs, inhibition of TAM recruitment, reprogramming of TAMs, targeting Toll-like receptors, inhibition of PI3K gamma, HDAC inhibitors, and inhibition of specific miRNA activities, and targeting TREM and exosomes). In addition, we present bioinformatic analyses that demonstrated that (i) TAM infiltration into many cancers correlated with poor survival (ii) the TAM infiltration was associated with the clinical stages of the cancer and (iii) there is a strong correlation between the TAM infiltrates and various immunosuppressive gene products. Although many clinical studies are underway to inhibit the immunosuppressive functions of TAMs through a variety of mechanisms, by either targeting TAMs alone or in combination with other therapeutics, we present various perspectives that need to be considered for the successful translational application of TAMs targeting alone or in combination with other therapies in the clinic.

Ex vivo blockade of PI3K gamma or delta signaling enhances the antitumor potency of adoptively transferred CD8+ T cells.

Adoptive T cell transfer therapy induces objective responses in patients with advanced malignancies. Despite these results, some individuals do not respond due to the generation of terminally differentiated T cells during the expansion protocol. As the gamma and delta catalytic subunits in the PI3K pathway are abundant in leukocytes and involved in cell activation, we posited that blocking both subunits ex vivo with the inhibitor IPI‐145 would prevent their differentiation, thereby increasing antitumor activity in vivo. However, IPI‐145 treatment generated a product with reduced antitumor activity. Instead, T cells inhibited of PI3Kγ (IPI‐549) or PI3Kδ (CAL‐101 or TGR‐1202) alone were more potent in vivo. While T cells coinhibited of PI3Kγ and PI3Kδ were less differentiated, they were functionally impaired, indicated by reduced production of effector cytokines after antigenic re‐encounter and decreased persistence in vivo. Human CAR T cells expanded with either a PI3Kγ or PI3Kδ inhibitor possessed a central memory phenotype compared to vehicle cohorts. We also found that PI3Kδ‐inhibited CARs lysed human tumors in vitro more effectively than PI3Kγ‐expanded or traditionally expanded CAR T cells. Our data imply that sole blockade of PI3Kγ or PI3Kδ generates T cells with remarkable antitumor properties, a discovery that has substantial clinical implications.

Abstract IA12: Targeting KRAS-regulated metabolic activities in pancreatic cancer

Abstract One promising direction for the development of therapeutic strategies for the treatment of KRAS-driven pancreatic ductal adenocarcinoma (PDAC) growth involves delineating the role of and mechanism for altered metabolism. KRAS-mutant PDAC is characterized by increased autophagy and macropinocytosis, processes that enhance nutrient availability that then supports the increased metabolic needs of PDAC. First, we addressed the role of KRAS in supporting autophagy-dependent PDAC growth. Surprisingly, we found that suppression of KRAS increased autophagic flux, as did pharmacologic inhibition of its effector ERK MAPK. We speculated that ERK inhibition might enhance PDAC dependence on autophagy by impairing KRAS-driven metabolic processes, and we demonstrate that either KRAS suppression or ERK inhibition decreased both glycolytic and mitochondrial functions. Furthermore, genetic or pharmacologic inhibition of autophagy synergistically enhanced ERK inhibitor-mediated antitumor activity. We conclude that concurrent inhibition of ERK and autophagy may be an effective PDAC treatment. Second, we evaluated mechanisms by which KRAS may drive macropinocytosis. These studies focused on KRAS G12R mutant PDAC. This mutation is rare in lung and colorectal cancers (~1%), yet high (~20%) in PDAC. We evaluated whether KRAS G12R is functionally distinct from the more common KRAS G12D/V mutant proteins. We found that KRAS G12D/V but not KRASG12R drives elevated macropinocytosis and that MYC is essential for this process in KRAS G12D/V but not KRAS G12R mutant PDAC. The KRAS G12R mutation uniquely disrupts interaction with a key effector, PI3K alpha, due to structural perturbations in the switch II region critical for effector interaction. Despite this defect, KRAS G12R-mutant PDAC displayed elevated macropinocytosis due to KRAS-independent PI3K gamma activity. Finally, the growth of KRAS G12R mutant PDAC in vitro and in vivo was preferentially sensitive to MEK-ERK inhibition compared to KRAS G12D/V. In summary, our elucidation of KRAS-dependent effector signaling mechanisms that drive altered metabolism identified novel therapeutic approaches for the treatment of PDAC. Citation Format: Kirsten L. Bryant, G. Aaron Hobbs, Channing J. Der. Targeting KRAS-regulated metabolic activities in 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 IA12.

A rearranged seco-steroid with new skeleton and five new abnormal progesteroids from Nodulisporium sp.

Abeo-nodulisporisteroid A (1), an abnormal steroid with a rearranged C3 chain at C-9 possibly derived from seco-ring A via Wagner-Meerwein rearrangement and oxidations, was obtained from Nodulisporium sp. together with five related new seco-progesteroids (nodulisporisteroids M-Q, 2–6), which were elucidated by the extensive spectroscopic analysis and X-ray crystallography. A plausible biosynthetic pathway of 1–6 was also proposed. Furthermore, these isolated compounds were also evaluated for cytotoxicities against five human tumour cell lines, antimicrobial activities, and inhibitions against PI3K gamma, Aβ42 aggregation, and α-glucosidase.

Abstract LB-B15: A novel PI3K gamma isoform selective small molecule kinase inhibitor demonstrates single agent anti-tumor activity and enhanced combination activity with checkpoint blockade in syngeneic mouse models of cancer

Abstract Immune checkpoint inhibitors have generated impressive clinical responses, however, a number of patients and cancer types remain resistant to checkpoint blockade. Immunosuppressive cells such as Tregs and MDSCs in the tumor microenvironment are hypothesized to mediate this resistance. The gamma (γ) isoform of PI3K has long been known to modulate myeloid and lymphocyte cell migration and function, including trafficking of monocytic and granulocytic cells into inflamed tissues such as tumors. Here we report the identification and characterization of BVD-723, a PI3Kγ selective small molecule kinase inhibitor which demonstrated single agent and enhanced combination anti-tumor activity[xx]with either an anti-PD-1 or anti-CTLA-4 antibody. In a mouse syngeneic colon cancer model,[xx]BVD-723 in combination with anti-PD-1 or anti-CTLA-4 resulted in complete regression[xx]of tumors in 35% (7/20) and 60% (12/20) of animals treated with each antibody respectively [versus 0% (0/20) and 5% (1/20) with either antibody alone]. Interestingly, the anti-tumor activity was coupled with a decrease in tumor infiltrating Tregs, granulocytic MDSCs, CD4+ T cells and an increase in the CD8+/Treg ratio, suggesting that inhibition of PI3Kγ by BVD-723 promotes immune-reactivation in the tumor microenvironment. Additionally, BVD-723 demonstrated potent synergy with an anti-PD-1 antibody in a syngeneic mouse model of lymphoma. Single agent BVD-723 activity was also observed in syngeneic mouse models of pancreas, colon, lymphoma and bladder cancers. Results from completed safety pharmacology, toxicology and in vivo efficacy studies support clinical evaluation of BVD-723[xx]as a monotherapy or in combination with the checkpoint inhibitors in cancer.[xx] Citation Format: Saurabh Saha, Linping Zhang, Thomas Hoock, Alex Aronov, Sudipta Mahajan, Michael Boyd, Jon Come, Veronique Damagnez, Wojciech Dworakowski, Suvarna Khare-Pandit, Arnaud LeTiran, Cameron Moody, Harwin O'Dowd, Joseph Prezioso, Setu Roday, Xiaoyan M. Zhang. A novel PI3K gamma isoform selective small molecule kinase inhibitor demonstrates single agent anti-tumor activity and enhanced combination activity with checkpoint blockade in syngeneic mouse models of cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B15.

Abstract IA22: Innate immune cell PI3K gamma as a target for suppression of pancreatic ductal adenocarcinoma

Abstract Ductal adenocarcinoma of the pancreas (PDAC) is a devastating disease that will afflict greater than 40,000 Americans. Less than 5% of these new pancreatic cancer patients will survive 5 years following diagnosis. PDAC is rarely detected in the early stages, with more than 80% of patients presenting with locally unresectable or metastatic disease at the time of diagnosis. As this form of cancer is also resistant to current cytotoxic therapies and ionizing radiation, novel therapeutic and diagnostic approaches are desperately needed to improve patient outcome. A characteristic feature of PDAC is the presence of an abundant, inflammatory infiltrate. In response to tumor-derived chemoattractants, we found that myeloid cells invade tumors where they promote immunosuppression, resulting in tumor growth and metastasis. We discovered that myeloid cell PI3-kinase gamma (PI3Kgamma) controls tumor immunosuppression, as PI3Kgamma signaling inhibits pro-inflammatory gene expression responses in macrophages, monocytes and granulocytes and promotes anti-inflammatory responses. Suppression of PI3Kgamma in mutant mice and in mice treated with pharmacologic inhibitors of PI3Kgamma promotes CD8+ T cell anti-tumor immune responses, leading to a 50% reduction in tumor growth and metastasis in orthotopic mouse models of pancreatic carcinoma and significant extension of survival in GEM models of PDAC (PDX1-cre; LSL-KrasG12D/+; LSL-Trp53 R172H/+). Furthermore, PI3Kgamma inhibition blocks macrophage stimulation of collagen deposition by fibroblasts in PDAC. We are exploring the combination of PI3kinase gamma inhibition with tumor cell and T cell targeted therapeutics could significantly reduce tumor growth and metastasis of PDAC. These approaches could improve patient outcomes by reducing tumor progression and preserving organ function by inhibiting desmoplasia. Citation Format: Megan Kaneda, Chanae Hardamon, Michael C. Schmid, Michael Bouvet, Franco Novelli, Emilio Hirsch, Andrew Lowy, Judith A. Varner. Innate immune cell PI3K gamma as a target for suppression of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA22.

Abstract PD5-2: Ph1b study of the PI3K inhibitor taselisib (GDC-0032) in combination with letrozole in patients with hormone receptor-positive advanced breast cancer

Abstract Background: Taselisib (GDC-0032) is a next-generation PI3K inhibitor with increased anti-tumor activity against PIK3CA mutant (MT) cancers. Taselisib is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30-fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that taselisib has enhanced activity against PI3K alpha isoform (PIK3CA) MT breast cancer cell lines and enhanced antitumor activity when combined with letrozole. Clinical data with single-agent taselisib also showed increased tumor shrinkage in patients with PIK3CA MT breast cancer as compared to patients with PIK3CA wildtype (WT) breast cancer. Material and Methods: A Phase 1b dose escalation study was conducted with evaluation of taselisib doses ranging from 6-9 mg QD in combination with letrozole 2.5mg QD in a modified 3+3 design. A dose expansion cohort was conducted with taselisib 6 mg QD. Safety and tolerability of GDC-0032 was assessed, as well as pharmacokinetics (PK), pharmacodynamic (PD) assessment by FDG-PET, and anti-tumor activity by RECIST. Results: As of 31 January 2014, 28 patients were enrolled onto this study with the completion of dose escalation and the dose expansion cohort. No dose limiting toxicities (DLTs) were observed at either the 6 mg (n = 20) or 9 mg (n = 8) dose levels. Adverse events (AEs) assessed by the investigator as related to taselisib in ≥10% of patients (any grade) included diarrhea, nausea, stomatitis, fatigue, rash, decreased appetite, hyperglycemia, dysgeusia, mucosal inflammation, vomiting, muscle spasms, asthenia, dry mouth, dry skin, pruritus, and aspartate aminotransferase increased. Grade 3 and 4 adverse events assessed by the investigator as drug-related and occurring in greater than one patient included diarrhea (14%), hyperglycemia (7%), and mucosal inflammation (7%). No apparent PK interactions were observed between taselisib and letrozole. The median number of prior systemic therapies was six, and promising efficacy data has been observed in these heavily pretreated patients. Metabolic partial responses via FDG-PET (≥ 20% decrease in mean SUVmax) were observed in 11 out of 18 patients assessed (61%). Confirmed partial responses by RECIST have been observed at both the 6mg and 9mg taselisib dose levels. For patients with measurable disease at baseline, the overall response rate of 38% was observed in patients with PIK3CA MT breast cancer and 9% in patients with PIK3CA WT breast cancer. Updated data on safety, PD, efficacy, and biomarker correlates will be presented. Conclusions: The combination of taselisib and letrozole is a well-tolerated regimen with promising preliminary efficacy in PIK3CA MT breast cancer patients. This preliminary Ph1b clinical data is consistent with taselisib preclinical and single-agent clinical data showing increased anti-tumor activity for taselisib in PIK3CA MT breast cancer as compared to PIK3CA WT breast cancer. Taselisib is being further investigated in the neoadjuvant setting in combination with letrozole in the LORELEI study in patients with untreated hormone receptor-positive breast cancer. Citation Format: Cristina Saura, Jasgit Sachdev, Manish R Patel, Andres Cervantes, Dejan Juric, Jeffrey R Infante, Donald Richards, Sandra Sanabria, Xuyang Lu, Joseph Ware, Timothy R Wilson, Hema Parmar, Jerry Y Hsu, Mafalda Oliveira, Eric P Winer, Daniel D Von Hoff, Jose Baselga, Ian E Krop. Ph1b study of the PI3K inhibitor taselisib (GDC-0032) in combination with letrozole in patients with hormone receptor-positive advanced breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD5-2.

Abstract PD1-3: Ph1b study of the PI3K inhibitor GDC-0032 in combination with fulvestrant in patients with hormone receptor-positive advanced breast cancer

Abstract Background: GDC-0032 is a next-generation PI3K inhibitor with increased anti-tumor activity against PIK3CA mutant cancers. GDC-0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30-fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that GDC-0032 has enhanced activity against PI3K alpha isoform (PIK3CA) mutant breast cancer cell lines. Preclinical data also show enhanced antitumor activity when GDC-0032 is combined with fulvestrant. Material and Methods: A Phase 1b dose escalation study was conducted with evaluation of GDC-0032 doses ranging from 6-9 mg QD in combination with fulvestrant 500mg q4wk (with loading dose of 500mg at day 1, 14 and 28) in a modified 3+3 design. A dose expansion cohort was conducted at the recommended Phase 2 dose of 6 mg QD. Safety and tolerability of GDC-0032 was assessed, as well as pharmacokinetics (PK), pharmacodynamic (PD) assessment of PI3K pathway inhibition by paired tumor biopsies and by FDG-PET, and anti-tumor activity by RECIST. Results: As of 1 Mar 2013, 17 patients were enrolled onto this study with the completion of dose escalation. No dose limiting toxicities (DLTs) were observed at either the 6 mg or 9 mg dose levels. Adverse events (AEs) assessed by the investigator as related to GDC-0032 in ≥10% of patients, were diarrhea, hyperglycemia, stomatitis, fatigue, asthenia, decreased appetite, nausea, mucosal inflammation and rash. No observed apparent PK interactions were observed between GDC-0032 and fulvestrant. The median number of prior systemic therapies was 6. Metabolic partial responses via FDG-PET (≥ 20% decrease in mSUVmax) were observed in 8 out of 11 patients assessed (73%). Confirmed partial responses by RECIST have been observed at both the 6mg and 9mg GDC-0032 dose levels. These include patients who have had prior treatment with fulvestrant. As of 29 May 2013, enrollment onto the dose escalation and expansion cohort has been completed (n = 27). Updated data on safety, pharmacodynamics, efficacy, and biomarker correlates will be presented. Conclusions: The combination of GDC-0032 and fulvestrant is a well-tolerated regimen with promising preliminary efficacy. GDC-0032 is being further investigated in combination with fulvestrant for patients with hormone receptor-positive advanced breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD1-3.

Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM.Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation.GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone.We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone.Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941.GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting.We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures:No relevant conflicts of interest to declare.

Abstract 4485: Preclinical characterization of PWT33597, a dual inhibitor of PI3-kinase alpha and mTOR

Abstract Phosphoinositide-3-kinase (PI3K) is an important mediator of tumor cell growth, survival and proliferation. In particular, PI3K alpha is important for signaling downstream of receptor tyrosine kinases and is also frequently amplified or mutationally activated in tumors, suggesting that selective inhibitors of this isoform may have therapeutic utility in the treatment of cancer. Downstream of PI3K, the mTOR kinase also plays a critical role in cellular growth and metabolism, and inhibitors of mTOR have demonstrated clinical benefit in several tumor types. We report here the discovery and characterization of PWT33597, a dual inhibitor of PI3K alpha and mTOR. PWT33597 inhibits PI3K alpha and mTOR in biochemical assays with IC50 values of 19 and 14 nM respectively, and is approximately 10-fold selective with respect to PI3K gamma and PI3K delta. Profiling of PWT33597 against 442 protein kinases (Ambit Kinomescan) revealed little or no cross-reactivity with either serine/threonine or tyrosine kinases, and there was little cross-reactivity with an additional panel of 64 pharmacologically relevant targets. In NCI-H460 and HCT116 tumor cells with mutationally activated PI3K alpha, PWT33597 inhibits phosphorylation of PI3K and mTOR pathway proteins with cellular IC50 values similar to its biochemical IC50 values. PWT33597 has good pharmacokinetic properties in multiple preclinical species, is not extensively metabolized in vivo and shows little potential for interaction with cytochrome P450 enzymes. Following a single oral dose in vivo, PWT33597 shows durable inhibition of PI3K and mTOR pathway signaling in xenograft tumors. High compound distribution into tumors and potent anti-tumor activity has been observed in multiple tumor xenograft models with activated PI3K/mTOR pathways. Also, administration of PWT33597 in mice is associated with transient increases in plasma insulin, consistent with an effect on PI3K/AKT signaling. A robust PK/PD relationship has been defined, which will guide interpretation of the planned phase I clinical study. IND-enabling studies with PWT33597 are currently in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4485. doi:10.1158/1538-7445.AM2011-4485

Autophagy Is a Key Myeloma Survival Pathway That Can Be Manipulated Therapeutically to Enhance Apoptosis

AbstractAbstract 4083In normal cells, autophagy is up-regulated under conditions of stress ensuring cell survival, suggesting that if it is inhibited cell death will ensue. In some cancer settings however it has been shown to enhance cell death. Therefore if autophagy is to be a target for anti-cancer therapy, it is important to determine whether to promote or inhibit the process. Cellular entry to autophagy can be manipulated via the PI3K/AKT/mTOR pathway, a pathway known to be important for myeloma cell growth and survival. In this study we investigated the effects of PI-103, a dual Class I PI3K and mTOR inhibitor, which activates autophagy, and used it as a tool to investigate the interaction of autophagy with other myeloma therapies. As the PI3K/AKT/mTOR pathway has been suggested to be a central pathway controlling entry into autophagy, we first determined the basal expression of key members of the pathway in a panel of myeloma cell lines. PI3K alpha, beta delta and gamma isoforms were expressed to varying degrees and constitutive activation of the pathway, (pAKT or pMTOR), was seen in the majority of cell lines. As predicted, treatment with PI-103 induced autophagy in myeloma cells as demonstrated by an increase in cellular inclusions staining positively with acridine orange, cleavage of the autophagosome marker, LC3, and a decrease in p62. PI-103 was shown to inhibit proliferation of all the myeloma cell lines and patient cells tested to varying degrees, although 100% growth inhibition was not seen. Bone marrow stromal cells were unaffected. The main mode of action of PI-103 was autophagy activation, and in keeping with this the extent of cell death measured by Annexin V/PI binding and trypan blue exclusion, was minimal. Cell cycle analysis demonstrated an increase in G0-G1 phase. The unfolded protein response (UPR) is important in myeloma cells and UPR activation and autophagy have been reported to be interlinked. Following exposure to PI-103, splicing of XBP1 mRNA to its active form, XBP1s, was seen, CHOP and ATF4 mRNA levels were also increased, consistent with activation of at least two branches of the UPR in response to PI3K/mTOR inhibition and autophagy induction. As myeloma cells activate autophagy as a pro-survival pathway following PI3-kinase inhibition, we were interested to understand the effect of blocking autophagy in this context. When PI-103 was combined with the autophagy inhibitor, Bafilomycin greatly enhanced apoptosis was seen. This increased apoptosis was seen in cells constitutively expressing p-AKT, with a complete loss of both phospho- and total levels of AKT and mTOR, an increase in the cleaved forms of caspase 3 and Bcl2, and massive activation of the IRE1 and PERK branches of the UPR. This effect was not seen in cells lacking p-AKT, a phenomenon described as ‘context-dependent oncogene addiction’ suggesting that measurement of p-AKT may be a useful predictive marker for response to joint PI3K/autophagy inhibition. Importantly the pro-apoptotic effects of the combination are not overcome by the presence of bone marrow cytokines, a more representative model of the physiological situation in vivo. In conclusion our data highlights the interplay between known myeloma growth and survival pathways and autophagy and suggests that combining PI3K inhibitors, with agents that target autophagy, may be beneficial for the treatment of myeloma, particularly in >50% of patients that express p-AKT. Disclosures:No relevant conflicts of interest to declare.

Chemical space sampling by different scoring functions and crystal structures

Virtual screening has become a popular tool to identify novel leads in the early phases of drug discovery. A variety of docking and scoring methods used in virtual screening have been the subject of active research in an effort to gauge limitations and articulate best practices. However, how to best utilize different scoring functions and various crystal structures, when available, is not yet well understood. In this work we use multiple crystal structures of PI3 K-gamma in both prospective and retrospective virtual screening experiments. Both Glide SP scoring and Prime MM-GBSA rescoring are utilized in the prospective and retrospective virtual screens, and consensus scoring is investigated in the retrospective virtual screening experiments. The results show that each of the different crystal structures that was used, samples a different chemical space, i.e. different chemotypes are prioritized by each structure. In addition, the different (re)scoring functions prioritize different chemotypes as well. Somewhat surprisingly, the Prime MM-GBSA scoring function generally gives lower enrichments than Glide SP. Finally we investigate the impact of different ligand preparation protocols on virtual screening enrichment factors. In summary, different crystal structures and different scoring functions are complementary to each other and allow for a wider variety of chemotypes to be considered for experimental follow-up.

Ablation of Phosphoinositide 3-Kinase-γ Reduces the Severity of Acute Pancreatitis

In pancreatic acini, the G-protein-activated phosphoinositide 3-kinase-gamma (PI3K gamma) regulates several key pathological responses to cholecystokinin hyperstimulation in vitro. Thus, using mice lacking PI3K gamma, we studied the function of this enzyme in vivo in two different models of acute pancreatitis. The disease was induced by supramaximal concentrations of cerulein and by feeding mice a choline-deficient/ethionine-supplemented diet. Although the secretive function of isolated pancreatic acini was identical in mutant and control samples, in both models, genetic ablation of PI3K gamma significantly reduced the extent of acinar cell injury/necrosis. In agreement with a protective role of apoptosis in pancreatitis, PI3K gamma-deficient pancreata showed an increased number of apoptotic acinar cells, as determined by terminal dUTP nick-end labeling and caspase-3 activity. In addition, neutrophil infiltration within the pancreatic tissue was also reduced, suggesting a dual action of PI3K gamma, both in the triggering events within acinar cells and in the subsequent neutrophil recruitment and activation. Finally, the lethality of the choline-deficient/ethionine-supplemented diet-induced pancreatitis was significantly reduced in mice lacking PI3K gamma. Our results thus suggest that inhibition of PI3K gamma may be of therapeutic value in acute pancreatitis.

Roles of PI3K in neutrophil function.

Neutrophils are terminally differentiated cells that play a vital role in host defense. It has recently become evident that phospholipid regulation plays an import role in many neutrophil functions. We review the regulation of neutrophil functions such as chemotaxis, superoxide production, and phagocytosis by phosphatidylinositol-3,4,5-trisphosphate (PIP3), which is generated in neutrophils by PI3K(gamma). Several lines of evidence are presented demonstrating the importance of this kinase in regulating chemotaxis, in particular the directionality of chemotactic migration. Evidence suggesting that this kinase is important for phagocytosis, especially during engulfment and the internalization of large particles, is also reviewed. Finally, it is suggested that PI3K is important for superoxide production and neutrophil priming. The common link between these seemingly diverse functions is that PI3K(gamma), via its phospholipid products, appears to be providing spatial-temporal cues for the binding of actin-organizing proteins.

CXCL16 Signals via Gi, Phosphatidylinositol 3-Kinase, Akt, IκB Kinase, and Nuclear Factor-κB and Induces Cell-Cell Adhesion and Aortic Smooth Muscle Cell Proliferation

CXCL16, a recently discovered transmembrane chemokine, is expressed in human aortic smooth muscle cell (ASMC). It facilitates uptake of low density lipoproteins by macrophages, resulting in foam cell formation. However, it is not known whether ASMC express CXCR6, the receptor for CXCL16, or whether CXCL16 affects ASMC biology. To dissect the biological and signal transduction pathways elicited by CXCL16, human aortic smooth muscle cells (HASMC) were treated with pharmacological inhibitors or transiently transfected with pathway-specific dominant-negative or kinase-dead expression vectors prior to the addition of CXCL16. HASMC expressed CXCR6 at basal conditions. Exposure of HASMC to CXCL16 increased NF-kappa B DNA binding activity, induced kappa B-driven luciferase activity, and up-regulated tumor necrosis factor-alpha expression in an NF-kappa B-dependent manner. However, treatment with pertussis toxin (G(i) inhibitor), wortmannin or LY294002 (phosphatidylinositol 3-kinase (PI3K inhibitors)), or Akt inhibitor or overexpression of dominant-negative (dn) PI3K gamma, dnPDK-1, kinase-dead (kd) Akt, kdIKK-beta, dnIKK-gamma, dnI kappa B-alpha, or dnI kappa B-beta significantly attenuated CXCL16-induced NF-kappa B activation. Furthermore, CXCL16 increased cell-cell adhesion and induced cellular proliferation in an NF-kappa B-dependent manner. In conclusion, CXCL16 is a potent and direct activator of NF-kappaB and induces kappa B-dependent proinflammatory gene transcription. CXCL16-mediated NF-kappa B activation occurred via heterotrimeric G proteins, PI3K, PDK-1, Akt, and I kappa B kinase (IKK). CXCL16 induced I kappa B phosphorylation and degradation. Most importantly, CXCL16 increased cell-cell adhesion and induced kappa B-dependent ASMC proliferation, indicating that CXCL16 may play an important role in the development and progression of atherosclerotic vascular disease.

Mouse beta 3a- and beta 3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways.

1. This study characterizes the mouse beta(3a)-adrenoceptor (AR) and the splice variant of the beta(3)-AR (beta(3b)-AR) expressed in Chinese hamster ovary cells (CHO-K1). 2. Stable clones with high (approximately 1200), medium (approximately 500) or low receptor expression (approximately 100 fmol mg protein(-1)) were determined by saturation binding with [(125)I]-(-)-cyanopindolol. Competition binding studies showed no significant differences in affinity of beta-AR ligands for either receptor. 3. Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The beta(3)-AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial beta-AR agonist CGP12177 and the beta-AR agonist (-)-isoprenaline caused concentration-dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration-dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. 4. PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the beta(3b)-AR but not in cells expressing the beta(3a)-AR at all levels of receptor expression. 5. CL316243 increased Erk1/2 phosphorylation with pEC(50) values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3K gamma inhibitor), PP2 (c-Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). 6. The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both beta(3a)- or beta(3b)-AR transfected cells. 7. These results suggest that in CHO-K1 cells, the beta(3b)-AR, can couple to both G(s) and G(i) to stimulate and inhibit cyclic AMP production respectively, while the beta(3a)-AR, couples solely to G(s) to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to G(i) or the generation of cyclic AMP.

Phosphopleckstrin Inhibits Gβγ-activable Platelet Phosphatidylinositol-4,5-bisphosphate 3-Kinase

Pleckstrin, the prototypic protein containing two copies of the pleckstrin homology domain, is a prominent substrate of protein kinase C in platelets and neutrophils. Both cell types have p85 subunit-containing phosphoinositide 3-kinase (p85/PI3K) and non-p85-containing PI3K (PI3Kgamma) that is activated by betagamma subunits of heterotrimeric GTP-binding proteins. We have shown that a PI3K product, phosphatidylinositol (PI) 3,4,5-trisphosphate, promotes pleckstrin phosphorylation in platelets. Since pleckstrin homology domains are thought to interact with Gbetagamma heterodimers and/or PI(4,5)P2, we have examined the effects of recombinant pleckstrins on platelet PI3Kgamma and p85/PI3K activities. Depending upon its phosphorylation/charged state, pleckstrin inhibits PI3Kgamma, but not p85/PI3K. Pleckstrin-mediated inhibition of PI3Kgamma is overcome by excess Gbetagamma and is restricted to PI(4,5)P2 as substrate, i.e. pleckstrin does not inhibit phosphorylation of PI()P or PI. Consistent with this, activation of protein kinase C by exposure of platelets to beta-phorbol diester (to increase endogenous pleckstrin phosphorylation) prior to platelet lysis causes inhibition of Gbetagamma-stimulatable PI3K activity only with respect to PI(4,5)P2 substrate. This phosphopleckstrin-mediated inhibition is overcome by increasing concentrations of Gbetagamma. We propose that phosphorylation of pleckstrin may constitute an important inhibitory mechanism for PI3Kgamma-mediated cell signaling.

Phosphoinositide 3-Kinase γ and p85/Phosphoinositide 3-Kinase in Platelets: RELATIVE ACTIVATION BY THROMBIN RECEPTOR OR β-PHORBOL MYRISTATE ACETATE AND ROLES IN PROMOTING THE LIGAND-BINDING FUNCTION OF αIIbβ3 INTEGRIN

Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-Kgamma. We now report that exposure of platelets to PKC-activating beta-phorbol myristate acetate (betaPMA) does not stimulate PI 3-Kgamma, but rather stimulates p85/PI 3-K, which associates with the cytoskeleton. Wortmannin is an inhibitor of both PI 3-Ks, known to act with more potency on p85/PI 3-K. betaPMA-stimulated 3-PPI accumulation is more sensitive to wortmannin (IC50 = 1.3 nM) than is SFLLRN- or thrombin-stimulated 3-PPI accumulation (IC50 = 10 nM). The activity of p85/PI 3-K in immunoprecipitates or in cytoskeletal fractions is inhibited more potently by exposure of platelets to wortmannin than is the activity of PI 3-Kgamma. betaPMA or SFLLRN promotes the conversion of platelet integrin alphaIIb/beta3 into a fibrinogen-binding form required for platelet aggregation. Activation of alphaIIb/beta3 in response to betaPMA or SFLLRN is inhibited by wortmannin with an IC50 of 1 nM in each case. Wortmannin inhibits neither activation of alphaIIb/beta3 by ligand-induced binding site antibody (anti-LIBS6 Fab) nor anti-LIBS6 Fab-induced platelet aggregation in the presence of fibrinogen, indicating that this type of "outside-in" signaling by alphaIIb/beta3 is largely PI 3-K-independent. We conclude that p85/PI 3-K, in preference to PI 3-Kgamma, contributes to activation of alphaIIb/beta3 when the thrombin receptor or PKC is stimulated.