Pan Phosphoinositide 3-kinase Research Articles

A Phase I Study of Copanlisib and Venetoclax in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a genomically heterogenous disease, which can be classified into distinct subgroups or clusters (Chapuy et al., Nat. Medicine, 2018, Wright et al., Cancer Cell, 2020). Leveraging knowledge of genetic alterations to design novel personalized treatment strategies may improve outcomes. Preclinical data suggest that the pan-phosphoinositide 3-kinase (PI3K) inhibitor, copanlisib, plus the BCL-2 inhibitor, venetoclax, are synergistic in genetically defined, high-risk, subtypes of DLBCL (Bojarczuk et al., Blood, 2019). We therefore hypothesized that copanlisib plus venetoclax would be an active treatment for patients with relapsed/refractory DLBCL, particularly in those with perturbed B-cell receptor (BCR)/PI3K signaling, which were predicted to be enriched in the relapsed setting. Here, we report the results of a phase I study of copanlisib plus venetoclax for patients (pts) with R/R DLBCL. Methods: This is an investigator-initiated, phase I, multicenter trial (NCT04572763) with the primary objective of determining the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of copanlisib and venetoclax in pts with R/R DLBCL. A standard 3+3 dose escalation design was employed, in which pts were treated with 60 mg IV copanlisib on days 1, 8, and 15 in addition to escalating doses of daily venetoclax for each 28-day cycle. During cycle 1, a venetoclax dose ramp-up was performed in the outpatient setting to a target dose of 200 mg (dose level (DL) +1), 400 mg (DL +2) and 800 mg (DL +3). The DLT observation period was the first cycle. Patients with progressive disease prior to the end of the DLT window were replaced. Key eligibility criteria included a confirmed diagnosis of DLBCL or high-grade B-cell lymphoma (HGBCL), relapse after or not a candidate for autologous stem cell transplant or chimeric antigen receptor (CAR) T-cell therapy, and adequate hematologic and organ function. Key exclusion criteria were poorly controlled hypertension or diabetes. CTCAE v5 and Lugano criteria were used to evaluate toxicity and efficacy, respectively. Results: As of July 21, 2023, 12 pts were evaluable. Patients were treated in DL +1 (n=5), DL +2 (n=3), and DL +3 (n=4). The median age was 62 years (range 24-87). Eighty three percent were male. Median number of prior treatments was 6 (range 1-11) and 10 pts had received prior CAR T-cell therapy. No DLTs were observed, though 2 pts in DL +3 were not able to escalate to 800 mg due to recurrent neutropenia and noncompliance, respectively. The most common toxicities were neutropenia (50%, all gr3/4), thrombocytopenia (42%, 8% ≥ gr3), nausea (33%, 8% ≥ gr3), fatigue (25%, 8% ≥ gr3), and headache (25%, 8% ≥ gr3) (Figure). Hyperglycemia occurred in 2 pts (both gr1). Serious adverse events occurred in 50% of pts and included fracture (n=1), hypoxia (n=2), and death (n=3), which were not related to study treatment. The deaths were related to progressive disease. Fifty percent of pts required drug holds (n=5 for venetoclax, n=5 for copanlisib). Sixty percent required growth factor support. There were no cases of laboratory or clinical tumor lysis syndrome. Sponsor support was withdrawn prior to completion of enrollment to DL+3, but DL+2 (copanlisib plus 400 mg venetoclax) was safe and well tolerated. The overall response rate (ORR) and complete metabolic response (CMR) rate was 8% (90% CI: 0 - 34%), as 1 pt in DL +1, who had received >5 lines of prior therapy, achieved a CMR, with response lasting 3 months. The median follow-up was 9.1 months. The median overall survival (OS) and progression-free survival (PFS) were 3.5 and 1.8 months, respectively. Conclusions: Copanlisib plus venetoclax is feasible and safe in pts with R/R DLBCL, with no DLTs observed. While it is possible that copanlisib plus venetoclax is active in select genomic subgroups of DLBCL, there was limited activity in this unselected, heavily pre-treated patient population.

Abstract B27: MTORC1/2 inhibition as a treatment strategy for PIK3CA mutant colorectal cancer

Abstract Colorectal cancer (CRC) is a leading cause of cancer-related death. PIK3CA mutations are common, leading to a constitutively active phosphoinositide-3 kinase (PI3K). An effective means to target this pathway has yet to be identified. We investigated the use of a panel of inhibitors targeting the PI3K pathway including copanlisib (dual PI3K/mTOR), BYL-719 (alpha isomer specific PI3K), GDC-0941 (pan PI3K), and TAK-228 (MTORC1/2). To test the efficacy of these inhibitors in CRC, murine organotypic cancer spheroids (MDOCS) were generated from the invasive adenocarcinomas of Apc and Pik3ca transgenic mice. These inhibitors were investigated at clinically relevant doses (100-400nM). Copanlisib and TAK-228 were the only inhibitors to result in a significant reduction in the size of the MDOCS (200nM; 27% p-value<0.001, 18% p-value<0.001, respectively). This result correlated with a decrease in the phosphorylation of AKT (ser473), RPS6, and 4EBP1. Minimal induction of apoptosis was observed using these inhibitors alone as measured by cleaved PARP and cleaved caspase 3. These results were confirmed in vivo using transgenic mice with TAK-228 (1mg/kg/day) and copanlisib (10mg/kg q2d x5) resulting in a reduction in lumen occlusion of the colon tumors. Persistent BCL-2 and BCL-xL signaling was hypothesized to be preventing the induction of apoptosis. To determine if inhibition of these BCL-2 family members would further sensitize these MDOCS to copanlisib and TAK-228, these inhibitors were tested in combination with navitoclax (ABT-263; BCL-2 family inhibitor). A dramatic enhanced sensitivity was observed in MDOCS (30% p-value<0.001, 23% p-value<0.001, respectively). This correlated with an induction of apoptosis as measured by cleaved caspase 3. Next a panel of eight CRC patient-derived organotypic cancer spheroids (PDOCS) were treated with the combination of TAK-228 and navitoclax. Differential sensitivity was observed across the panel (25% resistant, 37.5% intermediate, and 37.5% highly sensitive) owing to the importance of mutational profile with targeted therapies. These studies indicate the benefit of MTORC1/2 for the treatment of PIK3CA mutant CRC and with enhanced activity of the combination of MTORC1/2 inhibition in combination with BCL-2 family inhibition. These therapies deserve further investigation for the treatment of patients with PIK3CA mutant CRC. Citation Format: Rebecca A. DeStefanis, Susan N. Payne, Devon Miller, Cheri A. Pasch, Christopher Babiarz, Alyssa DeZeeuw, Stephanie L. Fricke, Carley Sprackling, Alexander E. Yueh, Demetra P. Korkos, Dana R. Van De Hey, Gioia Sha, Aurora Greane, Jeremy D. Kratz, Linda Clipson, Kristina A. Matkowskyj, Michael A. Newton, Dustin A. Deming. MTORC1/2 inhibition as a treatment strategy for PIK3CA mutant colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B27.

Brain-penetrant PQR620 mTOR and PQR530 PI3K/mTOR inhibitor reduce huntingtin levels in cell models of HD

One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds – the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.

Benefits versus risk profile of buparlisib for the treatment of breast cancer

ABSTRACTIntroduction: Activation of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathways occurs in 70% of breast cancer, including PIK3CA activating mutations, PTEN loss and AKT mutation. It is associated with poor prognosis and resistance to anti-HER2 and endocrine therapy. PI3K inhibitors are promising anticancer targets that can reverse resistance to these therapies. Buparlisib (BKM-120) is an orally active pan-PI3K inhibitor evaluated in different solid tumors as monotherapy or in combination.Areas covered: This article reviews preclinical data, clinical studies that have evaluated the efficacy and safety profiles of buparlisib as a monotherapy or in combination with targeted therapy (including endocrine and anti-HER2 therapy) or cytotoxics. The authors cover completed and ongoing studies to evaluate the benefit vs risk profile of buparlisib.Expert opinion: Targeting PI3K showed efficacy in BC. Buparlisib, a pan PI3K inhibitor, presents manageable but not negligible toxicity with an activity/toxicity ratio in favor of the use of emerging second generation, α–selective PI3K inhibitors for ongoing and future trials.

LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo

LY303511 was developed as a negative control of LY294002 without pan-phosphoinositide 3-kinase (PI3K) inhibition. We hypothesize LY303511 generate reactive oxygen species (ROS) to induce apoptosis for killing oral cancer cells. In MTS assay, LY303511 dose-responsively decreases survival in three kinds of oral cancer cells but little damage to normal oral cells (HGF-1). Two oral cancer cells (CAL 27 and SCC-9) with highly sensitivity to LY303511 were used. In 7-aminoactinomycin D (7AAD) assay, LY303511 slightly increases subG1 population in oral cancer cells. In annexin V/7AAD and/or pancaspase assays, LY303511 induces apoptosis in oral cancer cells but HGF-1 cells remains in basal level. In oxidative stress, LY303511 induces ROS and mitochondrial superoxide in oral cancer cells. In 8-oxo-2'-deoxyguanosine assay, LY303511 induces oxidative DNA damage in oral cancer cells. In zebrafish model, LY303511 inhibits CAL 27-xenografted tumor growth. Therefore, LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo.

Abstract PD5-12: Buparlisib (BUP) or placebo (PBO) plus fulvestrant (FUL) in postmenopausal patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC): Overall survival (OS) results from BELLE-2, a randomized, phase III study

Abstract Background: BELLE-2 (NCT01610284) met its primary endpoint of a statistically significant improvement in progression-free survival (PFS) with the pan-phosphoinositide 3-kinase (PI3K) inhibitor BUP+FUL compared with PBO+FUL in postmenopausal pts with HR+, HER2– ABC (Baselga et al. Lancet Oncol 2017). Here, we report the final results for OS. Methods: Postmenopausal women with HR+, HER2– ABC refractory to aromatase inhibitors were enrolled in this randomized, double-blind, placebo-controlled, Phase III study. Pts were randomized (1:1) to receive either oral BUP (100 mg/day) or PBO plus intramuscular FUL (500 mg per standard of care). Randomization was stratified by PI3K pathway status in archival tumor tissue (activated [PIK3CA mutation or loss of phosphatase and tensin homolog expression], non-activated or unknown) and visceral disease status (present or absent). Baseline PIK3CA mutation status in circulating tumor (ct) DNA was assessed in a subset of pts. The key secondary endpoint was OS, defined as time from randomization to date of death due to any cause, in the full population (PI3K pathway activated, non-activated and unknown), main study cohort (PI3K pathway activated and non-activated) and PI3K pathway-activated subpopulation. Other secondary endpoints included safety. OS by PIK3CA status in ctDNA was also investigated. Results: 1147 pts received BUP+FUL or PBO+FUL. In the full population and main cohort, OS results trended in favor of BUP+FUL vs PBO+FUL, but were not statistically significant (Table). Post-treatment use of antineoplastic medications was balanced between both arms. In pts with PI3K pathway-activated status, OS improved by approximately 6 months (Table). In pts with mutant PIK3CA ctDNA status, there was a slight trend in favor of BUP+FUL vs PBO+FUL (Table). Median OS, monthsHR (95% CI)P valueFull population N=1147BUP+FUL n=57633.20.87 (0.74–1.02)0.045* PBO+FUL n=57130.4 Main cohort n=851BUP+FUL n=42730.90.91 (0.75–1.09)0.144* PBO+FUL n=42428.9 PI3K pathway activated n=372BUP+FUL n=18833.60.81 (0.61–1.08)0.075** PBO+FUL n=18427.5 ctDNA PIK3CA mutant n=200BUP+FUL n=8726.00.81 (0.56–1.17)0.127** PBO+FUL n=11324.8 ctDNA PIK3CA non-mutant n=387BUP+FUL n=19931.41.12 (0.83–1.50)0.774** PBO+FUL n=18836.8 *One-sided P value. **Nominal P value. The safety profile of BUP+FUL was consistent with that previously reported, with no new safety concerns. Grade 3/4 adverse events with at least 5% difference between BUP+FUL vs PBO+FUL, respectively, were increased alanine aminotransferase (25.7% vs 1.1%), increased aspartate aminotransferase (18.0% vs 2.8%), hyperglycemia (15.4% vs 0.2%) and rash (8.0% vs 0.0%). Conclusions: The BELLE-2 OS results showed a trend in favor of BUP+FUL, but did not meet statistical significance. More selective PI3K inhibition may improve treatment benefit and safety compared with pan-PI3K inhibition. Further assessment of the predictive benefit of ctDNA-confirmed PIK3CA mutations in this setting is required. Citation Format: Campone M, Im S-A, Iwata H, Clemons M, Ito Y, Awada A, Chia S, Jagiełło-Gruszfeld A, Pistilli B, Tseng L-M, Hurvitz S, Masuda N, Cortés J, De Laurentiis M, Arteaga CL, Jiang Z, Jonat W, Sellami D, El-Hashimy M, Le Mouhaër S, Sankaran B, Bourdeau L, Baselga J. Buparlisib (BUP) or placebo (PBO) plus fulvestrant (FUL) in postmenopausal patients (pts) with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC): Overall survival (OS) results from BELLE-2, a randomized, phase III study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD5-12.

Will PI3K-targeted therapies for cancer become a reality?

In a recent issue of Annals of Oncology , Vuylsteke and colleagues presented the interim analysis of phase II PEGGY study on the use of pictilisib [a pan-phosphoinositide-3-kinase (PI3K) inhibitor] plus paclitaxel in hormone receptor positive human epidermal receptor 2 (HER2) negative (ER+/HER2−) locally advanced or metastatic breast cancer (1). This clinical trial is one among the many clinical clinicals to report clinical benefits and toxicity associated with PI3K-targeted therapies.

Better Understanding of Phosphoinositide 3-Kinase (PI3K) Pathways in Vasculature: Towards Precision Therapy Targeting Angiogenesis and Tumor Blood Supply.

The intracellular PI3K-AKT-mTOR pathway is involved in regulation of numerous important cell processes including cell growth, differentiation, and metabolism. The PI3Kα isoform has received particular attention as a novel molecular target in gene therapy, since this isoform plays critical roles in tumor progression and tumor blood flow and angiogenesis. However, the role of PI3Kα and other class I isoforms, i.e. PI3Kβ, γ, δ, in the regulation of vascular tone and regional blood flow are largely unknown. We used novel isoform-specific PI3K inhibitors and mice deficient in both PI3Kγ and PI3Kδ (Pik3cg(-/-)/Pik3cd(-/-)) to define the putative contribution of PI3K isoform(s) to arterial vasoconstriction. Wire myography was used to measure isometric contractions of isolated murine mesenteric arterial rings. Phenylephrine-dependent contractions were inhibited by the pan PI3K inhibitors wortmannin (100nM) and LY294002 (10µM). These vasoconstrictions were also inhibited by the PI3Kα isoform inhibitors A66 (10µM) and PI-103 (1µM), but not by the PI3Kβ isoform inhibitor TGX 221 (100nM). Pik3cg(-/-)/Pik3cd(-/-)-arteries showed normal vasoconstriction. We conclude that PI3Kα is an important downstream element in vasoconstrictor GPCR signaling, which contributes to arterial vasocontraction via α1-adrenergic receptors. Our results highlight a regulatory role of PI3Kα in the cardiovascular system, which widens the spectrum of gene therapy approaches targeting PI3Kα in cancer cells and tumor angiogenesis and regional blood flow.

PI-3K Inhibitors Preferentially Target CD15+ Cancer Stem Cell Population in SHH Driven Medulloblastoma.

Sonic hedgehog (SHH) medulloblastoma (MB) subtype is driven by a proliferative CD15+ tumor propagating cell (TPC), also considered in the literature as a putative cancer stem cell (CSC). Despite considerable research, much of the biology of this TPC remains unknown. We report evidence that phosphatase and tensin homolog (PTEN) and phosphoinositide 3-kinase (PI-3K) play a crucial role in the propagation, survival and potential response to therapy in this CD15+ CSC/TPC-driven malignant disease. Using the ND2-SmoA1 transgenic mouse model for MB, mouse genetics and patient-derived xenografts (PDXs), we demonstrate that the CD15+TPCs are 1) obligately required for SmoA1Tg-driven tumorigenicity 2) regulated by PTEN and PI-3K signaling 3) selectively sensitive to the cytotoxic effects of pan PI-3K inhibitors in vitro and in vivo but resistant to chemotherapy 4) in the SmoA1Tg mouse model are genomically similar to the SHH human MB subgroup. The results provide the first evidence that PTEN plays a role in MB TPC signaling and biology and that PI-3K inhibitors target and suppress the survival and proliferation of cells within the mouse and human CD15+ cancer stem cell compartment. In contrast, CD15+ TPCs are resistant to cisplatinum, temozolomide and the SHH inhibitor, NVP-LDE-225, agents currently used in treatment of medulloblastoma. These studies validate the therapeutic efficacy of pan PI-3K inhibitors in the treatment of CD15+ TPC dependent medulloblastoma and suggest a sequential combination of PI-3K inhibitors and chemotherapy will have augmented efficacy in the treatment of this disease.

The phosphoinositide 3-kinase signaling pathway is involved in the control of modified low-density lipoprotein uptake by human macrophages.

The transformation of macrophages into lipid-loaded foam cells is a critical early event in the pathogenesis of atherosclerosis. Both receptor-mediated uptake of modified LDL, mediated primarily by scavenger receptors-A (SR-A) and CD36 along with other proteins such as lipoprotein lipase (LPL), and macropinocytosis contribute to macrophage foam cell formation. The signaling pathways that are involved in the control of foam cell formation are not fully understood. In this study, we have investigated the role of phosphoinositide 3-kinase (PI3K) in relation to foam cell formation in human macrophages. The pan PI3K inhibitor LY294002 attenuated the uptake of modified LDL and macropinocytosis, as measured by Lucifer Yellow uptake, by human macrophages. In addition, the expression of SR-A, CD36 and LPL was attenuated by LY294002. The use of isoform-selective PI3K inhibitors showed that PI3K-β, -γ and -δ were all required for the expression of SR-A and CD36 whereas only PI3K-γ was necessary in the case of LPL. These studies reveal a pivotal role of PI3K in the control of macrophage foam cell formation and provide further evidence for their potential as therapeutic target against atherosclerosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11745-015-3993-0) contains supplementary material, which is available to authorized users.

Exploring a non-ATP pocket for potential allosteric modulation of PI3Kα.

Allosteric modulators offer a novel approach for kinase inhibition because they target less conserved binding sites compared to the active site; thus, higher selectivity may be obtained. PIK-108, a known pan phosphoinositide 3-kinase (PI3K) inhibitor, was recently detected to occupy a non-ATP binding site in the PI3Kα C-lobe. This newly identified pocket is located close to residue 1047, which is frequently mutated in human cancers (H1047R). In order to assess the interactions, stability, and any possible allosteric effects of this inhibitor on PI3Kα, extensive molecular dynamics (MD) simulations in aqueous solution were performed for the wild type (WT) human, WT murine, and H1047R human mutant PI3Kα proteins with PIK-108 placed in both catalytic and non-ATP sites. We verify the existence of the second binding site in the vicinity of the hotspot H1047R PI3Kα mutation through binding site identification and MD simulations. PIK-108 remains stable in both sites in all three variants throughout the course of the simulations. We demonstrate that the pose and interactions of PIK-108 in the catalytic site are similar in the murine WT and human mutant forms, while they are significantly different in the case of human WT PI3Kα protein. PIK-108 binding in the non-ATP pocket also differs significantly among the three variants. Finally, we examine whether the non-ATP binding site is implicated in PI3Kα allostery in terms of its communication with the active site using principal component analysis and perform in vitro experiments to verify our hypotheses.

Targeting the phosphoinositide 3-kinase pathway in hematologic malignancies

The phosphoinositide 3-kinase pathway represents an important anticancer target because it has been implicated in cancer cell growth, survival, and motility. Recent studies show that PI3K may also play a role in the development of resistance to currently available therapies. In a broad range of cancers, various components of the phosphoinositide 3-kinase signaling axis are genetically modified, and the pathway can be activated through many different mechanisms. The frequency of genetic alterations in the phosphoinositide 3-kinase pathway, coupled with the impact in oncogenesis and disease progression, make this signaling axis an attractive target in anticancer therapy. A better understanding of the critical function of the phosphoinositide 3-kinase pathway in leukemias and lymphomas has led to the clinical evaluation of novel rationally designed inhibitors in this setting. Three main categories of phosphoinositide 3-kinase inhibitors have been developed so far: agents that target phosphoinositide 3-kinase and mammalian target of rapamycin (dual inhibitors), pan-phosphoinositide 3-kinase inhibitors that target all class I isoforms, and isoform-specific inhibitors that selectively target the α, -β, -γ, or -δ isoforms. Emerging data highlight the promise of phosphoinositide 3-kinase inhibitors in combination with other therapies for the treatment of patients with hematologic malignancies. Further evaluation of phosphoinositide 3-kinase inhibitors in first-line or subsequent regimens may improve clinical outcomes. This article reviews the role of phosphoinositide 3-kinase signaling in hematologic malignancies and the potential clinical utility of inhibitors that target this pathway.

Characterization of the Mechanism of Action of the Pan Class I PI3K Inhibitor NVP-BKM120 across a Broad Range of Concentrations

The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused changes in expression of mitotic genes and the induction of a robust G(2)-M arrest. Tubulin polymerization assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynamics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the antitumor activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-α-dependent model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target activity start to manifest themselves at concentrations above 1 μmol/L in vitro and doses above 50 mg/kg in efficacy studies using subcutaneous tumor-bearing mice. Hence, careful concentration and dose range selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K.

Highlights of This Issue

Agents that target the Delta-like ligand 4 (Dll4)-Notch interaction have emerged as a new generation of molecules targeting tumor vasculature. Disrupting this interaction stimulates overdevelopment of the tumor vasculature, decreasing the effective blood supply to the tumor cells and restricting tumor growth. The work by Jenkins and colleagues describes an investigational human therapeutic antibody, MEDI0639, which binds a functional epitope on Dll4 that has not been described previously. Targeting this epitope results in potent modulation of endothelial cell function in vitro and modifies human vessels in vivo, establishing MEDI0639 as a new potential therapeutic to target Dll4.Glucose transporter 1 (Glut1) is upregulated in almost all cancer cells. By coupling chemical synthesis with glucose uptake and cell proliferation assays, Chen and his colleagues identified a Glut1 inhibitor, WZB117. WZB117 was shown to inhibit glycolysis and ATP synthesis, to block Glut1-mediated glucose transport, and to induce cell cycle arrest and senescence. Daily intraperitoneal injection of WZB117 led to significant reduction in the size of tumors grafted on nude mice. WZB117 and drugs cisplatin and paclitaxel show significant synergistic anticancer effects. All these indicate that Glut1 is a potential anticancer target and WZB117 is a candidate for developing anti-Glut1 anticancer drugs.Enhancer of Zeste Homolog 2 (EZH2) is upregulated in pancreatic cancer and has been associated with gemcitabine chemoresistance and poor prognosis. To explore its role as a novel therapeutic target, Avan and colleagues evaluated the interaction of the EZH2-inhibitor DZNeP with gemcitabine in pancreatic cancer cells, including primary cultures and spheroids growing in cancer stem cells selective medium. This study shows that DZNeP modulates EZH2/H3K27me3 expression and interacts synergistically with gemcitabine through reduction of CD133+ cells, enhancement of nucleoside transporters hENT1/hCNT1 expression, and inhibition of cell migration. These results provide a scientific rationale for future studies on this novel approach for treatment of pancreatic cancer.The pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120 was found, at high concentrations, to cause cell death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical profiling studies were used to identify the origin of these unexpected and apparently PI3K-independent effects. Brachmann and colleagues show that BKM120, at high concentrations, can act as a microtubule destabilizer via direct tubulin binding. Further analysis of clinical data, such as the assessment of mitotic markers in tumor biopsies from patients treated with BKM120, will be required to fully confirm that the compound off-target activity is not clinically relevant.

Discovery and Optimization of New Benzimidazole- and Benzoxazole-Pyrimidone Selective PI3Kβ Inhibitors for the Treatment of Phosphatase and TENsin homologue (PTEN)-Deficient Cancers

Most of the phosphoinositide-3 kinase (PI3K) kinase inhibitors currently in clinical trials for cancer treatment exhibit pan PI3K isoform profiles. Single PI3K isoforms differentially control tumorigenesis, and PI3Kβ has emerged as the isoform involved in the tumorigenicity of PTEN-deficient tumors. Herein we describe the discovery and optimization of a new series of benzimidazole- and benzoxazole-pyrimidones as small molecular mass PI3Kβ-selective inhibitors. Starting with compound 5 obtained from a one-pot reaction via a novel intermediate 1, medicinal chemistry optimization led to the discovery of compound 8, which showed a significant activity and selectivity for PI3Kβ and adequate in vitro pharmacokinetic properties. The X-ray costructure of compound 8 in PI3Kδ showed key interactions and structural features supporting the observed PI3Kβ isoform selectivity. Compound 8 achieved sustained target modulation and tumor growth delay at well tolerated doses when administered orally to SCID mice implanted with PTEN-deficient human tumor xenografts.

Abstract 3741: Inhibition of PI3Kα kinase by a selective small molecule inhibitor suppresses B-cell proliferation and leukemic cell growth

Abstract Background: Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellular lipid kinases that phosphorylate the 3 position hydroxyl group of the inositol ring of phosphotidylinositol. The PI3K pathway is frequently activated in human cancers and thus represents an attractive target for small molecule inhibitors. Pan-PI3K inhibitors currently in development have been associated with adverse side-effects such as insulin resistance, thus necessitating the need to develop isoform specific inhibitors of PI3K. Herein, we describe the biological and pharmacokinetic properties of RP5264, a small molecule PI3Kα inhibitors with scope to be further developed as a clinical candidate for hematological malignancies mediated by B-cells. Methods: Activity of RP5264 on individual PI3K isoforms was determined by a Homogenous Time Resolved Fluorescence assay (Millipore, Billerica, MA) with modifications. Cell based selectivity assays against β, α, or ≤ isoforms was assessed by testing the effect of the compound on PDGF, LPA, or c5a induced Akt phosphorylation in NIH-3T3 or RAW cells. Similarly, inhibition of cellular PI3Kα activity was determined in an IgM induced human B-cell proliferation as well as LPS-induced CD19 assays. Ability to arrest cell growth and induce apoptosis was also tested. Viability assays was conducted to determine the growth inhibitory effect of the compounds in leukemic cells. Pharmacokinetic behaviour of compounds in plasma after single dose oral administration was determined in female Balb/c mice. Results: RP5264 inhibited PI3Kα activity in enzyme and cell based assays with IC50 and EC50 values of 22.2 & 24.3 nM respectively. The compound displayed a high degree of selectivity over the alpha (>1000 fold), beta (>30-50 fold), and gamma (>15-50 fold) isoforms. Additionally, the compound caused a half-maximal inhibition of human whole blood CD19 cell proliferation between 100-300 nM. Treatment of PBMC with RP5264 resulted initially in a G2/M arrest followed by subsequent increase in the number of Sub G0 cells. Viability assays demonstrated that the compound caused a significant inhibition in growth as well as Akt phosphorylation of immortalized and primary leukemic cells. Further, the compound exhibited good oral absorption with favourable pharmacokinetic properties in rodents. Conclusions: Results demonstrate the PI3K delta selective nature of RP5264 along with an ability to suppress proliferation and Akt phosphorylation in cancer cells. In vitro selectivity and potency data indicate the therapeutics potential of the compounds in hematological cancers without the deleterious effects commonly associated with the Pan PI3K inhibitors. RP5264 is poised to enter clinical development in 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3741. doi:1538-7445.AM2012-3741

Abstract B155: A phase I study of the MEK inhibitor GSK1120212 (GSK212) in combination with PI3K/mTOR inhibitor GSK2126458 (GSK458) in patients with advanced solid tumors.

Abstract Background: This is a Phase I, open-label, dose escalation, pharmacokinetic (PK), pharmacodynamic (PD) and safety study in patients (pts) with advanced solid tumors exploring the combination of the investigational drugs GSK458 and GSK212. GSK212 is a reversible, highly selective, allosteric inhibitor of mitogen-activated extracellular signal regulated kinases 1 and 2 (MEK1 and MEK2). GSK458 is a potent, reversible, ATP-competitive, pan-phosphoinositide-3 kinase (PI3K) inhibitor, which also inhibits the mammalian target of rapamycin (mTOR), and the common activating mutations of catalytic isoform p110α. The MTD for daily, continuous dosing of monotherapy GSK458 is 2.5mg and GSK212 is 2.0mg. Methods: The study consists of two parts: dose escalation in Part 1 (ongoing) uses a zone design (3+3 rules) to study safety, tolerability, PK, PD and clinical activity, and to establish the maximum tolerated doses (MTDs) and schedules for the combination. Adult pts with relapsed or refractory solid tumors are receiving oral doses of GSK458+GSK 212 once daily until disease progression or toxicity. Efficacy is assessed using RECIST 1.1. Part 2 will examine safe dose combinations from Part 1 in patients with tumors likely to respond to MEK/PI3K pathway inhibition. Genetic and protein profiles collected from blood and tumor samples will be used to explore relationships between predictive biomarkers of response and efficacy. Results: 34 pts(45% female) with a mean age of 58y (range 37–82y) have enrolled. Safety data includes31 subjects who have received ≥1 dose of GSK458+GSK212. Most commonly reported drug-related AEs >10%, n=31) include rash (68%; G3/4, 3%), diarrhea (42%; G3/4, 6%), nausea (29%; G3/4, 0%), vomiting (23%; G3/4, 3%), decreased appetite (19%; G3/4, 0%), stomatitis (13%; G3/4, 3%) and fatigue (13%; G3/4; 3%). Of 21 serious adverse events (SAEs) reported in 14 pts, 3 were considered related to study drug. Stable disease has been observed in several patients with the longest being at 6 months (renal cell carcinoma and colorectal cancer). The combination of GSK458 0.5 mg + GSK212 2.0 mg exceeded the MTD. However, once daily and continuous combination dosing of GSK458 (0.5, 1.0 or 1.5mg) + GSK212 ≤ 2.0 mg were well tolerated. Conclusions: The preliminary safety profile and anti-tumor activity of this combination warrants further investigation in continued dose escalation and planned expansion cohorts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B155.

Abstract 4489: Preclinical profile of novel, potent, and selective PI3K delta inhibitors

Abstract Background: Phosphoinositide-3 kinase (PI3K) belongs to a class of intracellular lipid kinases that phosphorylate the 3 position hydroxyl group of the inositol ring of phosphotidylinositol. The PI3K pathway is frequently activated in human cancers and thus represents an attractive target for small molecule inhibitors. Pan-PI3K inhibitors currently in development have been associated with adverse side-effects such as insulin resistance, thus necessitating the need to develop isoform specific inhibitors of PI3K. Herein, we describe the biological and pharmacokinetic properties of representative molecules from a series of novel and small molecule PI3KΔ inhibitors with scope to be further developed as clinical candidates for hematological malignancies such as acute myeloid leukemia and Non-Hodkin's lymphoma. Methods: Activity of test compounds on individual PI3K isoforms was determined by a Homogenous Time Resolved Fluorescence assay (Millipore, Billerica, MA) with modifications. Cell based selectivity assays against α, β, or γ isoforms was assessed by testing the effect of the compounds on PDGF, LPA, or c5a induced Akt phosphorylation in NIH-3T3 or RAW cells. Similarly, inhibition of cellular PI3KΔ activity was determined in a IgM induced human B-cell proliferation assay. Cell viability assay was conducted to determine the growth inhibitory effect of the compounds in cell lines representative of human acute and chronic leukaemia and lymphoma. Pharmacokinetic behaviour of compounds in plasma after single dose oral administration was determined in female Balb/c mice. Results: Among the compounds evaluated, RP5175 and RP5182 inhibited PI3KΔ activity in enzyme and cell based assays with IC50 values of 9.6 & 37.3 nM and EC50 values of 7.6 & 18.9 nM respectively. Besides, the compounds displayed a high degree of selectivity over the alpha (>500 fold), beta (>400 fold), and gamma (>15-40 fold) isoforms. Viability assays demonstrated that the compounds caused a significant inhibition in growth of THP-1, TOLEDO, and HL-60 cells. Pharmacokinetic studies in female Balb/c mice indicated good oral absorption with favourable peak plasma concentrations. Conclusions: Results demonstrate the PI3K delta selective nature of RP5175 and 5182 along with an ability to suppress proliferation of cancer cells. In vitro selectivity and potency data indicate the therapeutics potential of the compounds in hematological cancers without the deleterious effects commonly associated with the Pan PI3K inhibitors. The compounds are currently being tested for in vitro and in vivo efficacy across various cancer cell lines and xenograft models besides selectivity against other kinases. 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 4489. doi:10.1158/1538-7445.AM2011-4489

Small Molecule SHIP Agonist AQX-MN100 Inhibits Multiple Myeloma (MM) Cell Growth In NOD/SCID Mice and Reduces Immune Suppressive Myeloid Derived Suppressor Cells (MDSC) and Regulatory T Cells (Tregs) In Mouse Model

AbstractAbstract 792We recently described a novel anti-MM drug (AQX-MN100) which is a small molecule agonist of SHIP (Src homology-2 (SH2) containing inositol-5¢-phosphatase) a signaling molecule found only in hemopoietic cells.(Ong et al, Blood; 110:1942, 2007) The molecule was developed using a high-throughput SHIP enzyme assay to screen an invertebrate marine natural product library and isolate the Pelorol.(Yang et al Org Lett; 7:1073, 2005) SHIP normally functions to negatively regulate the PI3K pathway important to normal hemopoietic cells growth and function. Inappropriate activation of the phosphoinositide 3- kinase (PI3K) pathway has been shown to be involved in the pathogenesis of MM and tumour aggressiveness correlates with the degree of activation. The critical role the PI3K/Akt signaling pathway plays in regulating MM cell survival, has stimulated efforts in designing therapeutics that target this pathway. Pan PI3K inhibitors have limited utility in a clinical setting because of their inhibitory effects on all isoforms of the PI3K family as well as non-PI3K targets. SHIP is an exceptionally good target for MM and other hematopoietic disorders that display elevated PI3K/Akt signaling because its expression is restricted to hemopoietic cells. We have shown that an analogue of Pelorol, AQX-MN100 is able to inhibit PI3K signaling and prevent phosphorylation of Akt. AQX-MN100 induced MM cell line apoptosis mediated by caspase and was specific for SHIP expressing cells which are exclusively hematopoietic. AQX-MN100 also enhances the growth inhibition effects of current myeloma drugs Dexamethasone and Bortezomib on human MM tumour cell lines in vitro. (Kennah et al Expt Hematol; 37:1274, 2009)In this study we have extended these finding to further evaluate the role of this compound in the treatment of myeloma. NOD-SCID mice were injected in the lateral flanks with 2 million luciferase tagged MM1.S multiple myeloma cells in Matrigel. Tumors were allowed to establish for two weeks and then either AQX-MN100 or vehicle was administered in an oil deposit subcutaneously in the lower flank at a dose of 50 mg/kg every three days. Tumor volume was quantified by imaging on a Xenogen IVIS 200 after 6 and 11 days. These studies demonstrate a significant reduction of tumor volume at 6 days p<0.05 and a highly significant reduction at 11 days p<0.01 in the mice receiving AQX-MN100 as compared to vehicle.We have shown that AQX-MN100 can directly kill MM cells in in vitro and in vivo. However, based on the known functions of SHIP, we predict that SHIP agonists will additionally target critical steps in MM pathogenesis in vivo, including the ability of MM cells to interact with stromal elements and to subvert the immune system. In order to evaluate this later feature we evaluated the ability of SHIP agonists to reverse the tumor associated immune suppression in MM patients. Tumor and host cell/tumor microenvironment secreted factors promote the production and activation of cells associated with cancer progression: the immune suppressive myeloid derived suppressor cells (MDSC) and regulatory T cells (Tregs). These cells normally regulate immune responses by inhibiting the activation of immune effector cells. The involvement of SHIP in the regulation of these cells is predicted by the observation that MDSC and Treg numbers are elevated in SHIP deficient mice. In this study Balb/C mice, 6 mice/group in duplicate were given either AQX-MN100 3 mg/kg and 10 mg/kg or vehicle once daily orally. At the end of three weeks mesenteric lymph nodes were harvested and subjected to FACS analysis to determine the proportion of MDSC (CD11b+Gr1+) and Treg (CD4+CD25+FoxP3+) cells. Spleen cells were also analysed for B cells, NK cells and granulocytes. In both of the AQX-MN100 treated groups the numbers of MDSC and Tregs were significantly lower than controls while Total CD11b, Total CD3, and spleen B, NK and granulocytes were not different from vehicle treated controls.The known role of SHIP in regulating hemopoietic cell function and the role of SHIP agonists in MM cell killing as well as additional actions on other aspects of MM pathophysiology may make them a powerful treatment option for MM, either alone or in synergy with other known MM therapies. Further development of this agent for the treatment of MM is ongoing. Disclosures:Mui:Aquinox: Equity Ownership, Patents & Royalties. Ong:Aquinox: Equity Ownership, Patents & Royalties. Anderson:Aquinox: Equity Ownership, Patents & Royalties. Sutherland:Celgene: Honoraria; Orthobiotech: Honoraria.

A phase I study evaluating the pharmacokinetics (PK) and pharmacodynamics (PD) of the oral pan-phosphoinositide-3 kinase (PI3K) inhibitor GDC-0941.

2613 Background: The PI3K-PTEN-AKT signaling pathway is deregulated in a wide variety of cancers. GDC-0941 is a potent and selective oral pan-inhibitor of class I PI3K with 3 nM IC50 for the p110-alpha subunit in vitro and demonstrates activity in preclinical xenograft models (breast, ovarian, lung, and prostate). Material and Methods: Patients (pts) with histologically confirmed advanced solid tumors and performance status 0–1 were enrolled in a phase I study (GDC4254g) of GDC-0941 using a 3+3 escalation design. Treatment was a single dose of GDC-0941 with a 1-week washout, followed by GDC-0941 QD on a 3-wk on, 1-wk off schedule. Phase I objectives included an evaluation of PD endpoints in surrogate tissue (pAKT in platelet-rich plasma [PRP]) and tumor tissue (pS6 in paired tumor biopsies) and FDG-PET. Results: Twenty-six pts have been enrolled in 7 successive cohorts (15–130 mg QD). GDC- 0941 was generally well tolerated with no DLT observed to date. The most frequently occurring drug-related adverse events (AEs) have been grade 1–2 nausea, diarrhea, vomiting, fatigue, and dysgeusia. There has been one drug-related grade 4 AE of hyperglycemia in a cholangiocarcinoma pt at the 130 mg dose level during cycle 2 in the setting of concomitant steroid use. GDC-0941 displays dose-proportional increases in mean Cmax and AUCinf. Decreases in pS6 staining of >50% have been observed in paired tumor biopsies in addition to decreases of >90% in pAKT levels assayed in PRP from pts treated at 80 mg and higher. Signs of biologic activity have been observed in 3 pts (ovarian cancer, triple negative breast cancer, and ocular melanoma) treated at ≥100 mg GDC-0941 with reductions (≥30% in mean SUVmax) in tumor FDG avidity observed on PET scan and an ∼80% decrease in CA-125 in the ovarian cancer pt, who remained on-study for ∼5 months. Conclusions: GDC-0941 is generally well tolerated at 15 to 130 mg QD. Decreases in pAKT levels in PRP and decreases in pS6 staining in paired tumor biopsies are consistent with downstream modulation of the PI3K pathway. FDG-PET reductions have been observed along with a CA-125 decrease in a pt with ovarian cancer. Dose-escalation continues and updated PK/PD data will be presented. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Genentech, Institute of Cancer Research Arno Therapeutics, AstraZeneca, Exelixis, Genentech, GlaxoSmithKline, Merck, Novartis, Pfizer Roche Arno Therapeutics, AstraZeneca, Exelixis, Genentech, GlaxoSmithKline, Merck, Novartis, Pfizer Genentech