Selective PI3Kδ Inhibitor Research Articles

Results of the Mantle Cell Lymphoma Subset from a Phase 2a Study of Copanlisib, a Novel PI3K Inhibitor, in Patients with Indolent and Aggressive Lymphoma

Introduction: Mantle cell lymphoma (MCL) is an aggressive disease that is incurable with conventional therapy and the outcome of which remains the poorest amongst B-cell lymphomas. Phosphoinositide-3 kinase (PI3K) pathway activation contributes to MCL pathogenesis, but early-phase studies of the PI3K-δ selective inhibitor idelalisib have reported lower responses in MCL compared with indolent non-Hodgkin lymphoma (NHL) subtypes (Kahl et al., Blood 123:3398-405, 2014; Gopal et al., NEJM 370:1008-18, 2014). In addition, although PI3K-δ is highly expressed in MCL, PI3K-α shows wide variation and expression increases with relapse (Iyengar et al., Blood 121;2274-84, 2013). Copanlisib is a novel pan-Class I PI3K inhibitor with potent preclinical inhibitory activity against both PI3K-α and PI3K-δ isoforms. Preliminary results from a phase 2a study of copanlisib in patients with relapsed/refractory NHL or chronic lymphocytic leukemia (CLL) have been reported (Dreyling et al., ASH 2013), with an expansion cohort in patients with aggressive lymphoma ongoing. We report here the final results of the MCL subset.Methods: Patients with histologically confirmed indolent and aggressive NHL and relapsed after, or refractory to, ≥2 prior lines of treatment were eligible. Copanlisib was administered at the starting dose of 0.8 mg/kg as a 1 hour intravenous infusion on days 1, 8 and 15 of a 28-day cycle until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR) as assessed per independent radiologic review according to the response criteria for lymphoma (Cheson et al., JCO 17:1244, 1999). Secondary endpoints included progression-free survival (PFS) and duration of response (DOR), safety and tolerability.Results: As of February 28, 2015, of the 81 patients enrolled, a total of 11 patients with MCL were treated. Median age was 70 years (range 60-85), M/F= 8/3. The median number of prior lines of treatment was 3 (range 3-9) with a median time of 1.4 months since last systemic anti-cancer therapy. All patients previously received rituximab and 8 patients (73%) were refractory to the last therapy. The median duration of treatment was 17 weeks (range 3-59), corresponding to a median number of 4 cycles (range 1-15), with a close adherence to planned dose (median 92%). The most common drug-related adverse events (AEs) of all grades were hyperglycemia (8 patients, 73%), hypertension (6 patients, 55%), neutropenia (5 patients, 46%) and fatigue (4 patients, 36%). Grade 3-4 AEs occurring in 2 or more patients included: neutropenia (2 patients with grade 3, 18%; 3 patients with grade 4, 27%), hypertension (3 patients with grade 3, 27%), hyperglycemia (2 patients with grade 3, 18%), and fatigue (2 patients with grade 3, 18%). One grade 5 AE of acute respiratory failure, was assessed as drug-related by the investigator. Dose reductions and delays possibly due to study drug-related adverse events (AE) were reported in 2 (18%) and 6 (55%) patients, respectively. Two patients were discontinued from study treatment due to adverse events (lung infection and non-melanoma skin cancer). All 11 patients were included into efficacy assessment, although one clinical PD was not confirmed by radiologic measurement. The ORR as determined by independent radiologic review was 64% (2 CRu and 5 PRs). The median duration of response was 150 days (95% CI: 56, 434); 33% of responders had a DOR of at least 270 days. PFS ranged from 7 to 547 days [median 112 days (95% CI: 42, 377)].Conclusions: Copanlisib was active as a single-agent, with an ORR of 64%, and had a manageable safety profile in patients with heavily pretreated, advanced refractory/relapsed MCL. These results support the potential role of inhibiting both PI3k-α and PI3K-δ in relapsed MCL. Based on these results, a phase 2 trial of copanlisib in ibrutinib-pretreated patients with MCL is under way (NCT02455297). DisclosuresCunningham:Astra Zeneca: Research Funding; Merrimack: Research Funding; Amgen: Research Funding; Celgene: Research Funding; Merck Serono: Research Funding; Sanofi: Research Funding; Medimmune: Research Funding; Bayer HealthCare Pharmaceuticals: Research Funding. Zinzani:Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; J&J: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Assouline:Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy. Mollica:Pfizer: Consultancy; BMS: Consultancy; Novartis: Consultancy. Giurescu:Bayer Pharma AG: Employment. Gorbatchevsky:Bayer HealthCare Pharmaceuticals: Employment. Neves:Bayer HealthCare: Employment. Lemos:Bayer HealthCare: Employment. Grunert:Bayer Pharma AG: Employment. Hiemeyer:Bayer Pharma AG: Employment. Childs:Bayer HealthCare Pharmaceuticals: Employment.

Exposure-Response of Idelalisib Administered in Combination with Ofatumumab for the Treatment of Relapsed Chronic Lymphocytic Leukemia

Abstract Background: Idelalisib (Zydelig®) is a potent and selective PI3Kδ inhibitor approved for the treatment of relapsed CLL (in combination with rituximab) and relapsed iNHL subtypes, FL and SLL. Relationship between plasma exposures to idelalisib or its inactive circulating metabolite, GS-563117, and clinical efficacy and safety were evaluated in relapsed/refractory CLL subjects following administration of idelalisib 150 mg twice daily (BID) in combination with ofatumumab (Study GS-US-312-0119). Methods: Idelalisib and GS-563117 plasma exposures (Cmax, AUCtau and Ctau) were generated using population pharmacokinetic models based on data from several phase 1/2/3 clinical studies. Efficacy and safety data from the Phase 3, randomized, open-label study (GS-US-312-0119) were included in the PK/PD analyses. Efficacy endpoints including best overall response rate (BOR), duration of response (DOR), progression -free survival (PFS), sum of products of the greatest perpendicular diameters (SPD) of index lesions, and lymph node response rate (LNR) were evaluated against idelalisib plasma exposure (AUCtau and Ctau). Safety endpoints evaluated included ≥ Grade 3 neutropenia, skin rash, infection, pneumonia, pneumonitis, diarrhea, colitis, and aspartate aminotransferase (AST) or alanine aminotransferase (ALT) elevation versus idelalisib and GS-563117 plasma exposure (AUCtau and Cmax). Kaplan-Meier survival analysis, Cox proportional hazard model/Logistic regression analyses were conducted for key efficacy and safety endpoints. Results: Median idelalisib exposures were similar between responders (complete response [CR] or partial response [PR]) versus nonresponders (stable disease [SD] or progressive disease [PD]) and across the 4 response categories (p≥0.67; Table 1). As idelalisib exposure increased over quartiles, there was no clear trend of prolonged DOR (p≥0.49), PFS (p≥0.15) or change in SPD. No relationship between LNR (responder versus non-responder) and idelalisib exposures was observed. No association was observed between idelalisib or GS-563117 plasma exposure vs incidence of neutropenia, diarrhea (p≥0.50), skin rash, infection (p≥0.18), pneumonia (p≥0.38), pneumonitis (p≥0.20), colitis (p≥0.43), and aspartate aminotransferase (AST) or alanine aminotransferase (ALT) elevation. Conclusions: No exposure-response relationships were observed for efficacy or safety endpoints at idelalisib 150 mg BID. These results are consistent with previous findings of exposure to idelalisib in subjects with CLL (in combination with rituximab) and iNHL and support idelalisib 150 mg BID in combination with ofatumumab for the treatment of CLL. Table 1. Summary of idelalisib Ctau in Study GS-US-312-0119 based on BOR category/status BOR Category BOR Status Responder Non-responder CR PR SD PD N 131 40 1 130 30 1 Median (ng/mL) 353 377 298 353 338 583 Q1, Q3 (ng/mL) 229, 518 268, 504 298, 298 229, 518 233, 433 583, 583 Min, Max (ng/mL) 76.2, 1529 62, 1184 298, 298 76.2, 1529 62, 1184 583, 583 CR: complete responder, PR: partial responder, SD: stable disease, PD: progressive disease. CR and PR were responder; SD and PD were non-responder. Disclosures Sharma: Gilead Sciences: Employment, Equity Ownership. Guo:Gilead Sciences: Employment, Equity Ownership. Jin:Gilead Sciences: Employment, Equity Ownership. Li:Gilead Sciences: Employment, Equity Ownership. Dubowy:Gilead Sciences: Employment, Equity Ownership. Newcomb:Gilead: Employment. Ramanathan:Gilead Sciences: Employment, Equity Ownership.

Abstract C103: The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models

Abstract Understanding the in vivo responses to immunoregulatory agents provides a basis for building more efficacious combination regimens. Pharmacologic inhibition of the oncogenic PI3Kδ pathway has been shown to be active in patients with hematopoietic malignancies. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, through the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses, suggesting that it may have additional utility in this patient population. We identified a similar immunomodulatory role for the PI3Kδ-selective inhibitor INCB050465 in a preclinical model of pancreatic cancer, where an increase in the number of CD8+ T cells, a decrease in the number of suppressor cells and efficacy were seen. Therefore we explored the potential of INCB050465 in additional preclinical solid tumor models, alone and in combination with other immunotherapeutic agents. INCB050465 inhibited tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition was not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate immune-mediated mechanisms, tumors were analyzed for modulation of gene expression and immune phenotype after mice received short-term treatment. INCB050465 was shown to significantly downregulate the T cell gene signature in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. As seen previously, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T-cell-inflamed model by reducing both Treg and M2 macrophages, which promotes re-activation of both CD4+ and CD8+ T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell response over time. In summary, pharmacological inhibition of PI3Kδ can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and complex cellular responses may provide rationale to identify individuals who may benefit from PI3Kδ inhibitor-based immunotherapy combinations in the clinic. Citation Format: Holly K. Koblish, Liang-Chuan Wang, Michael Hansbury, Yue Zhang, Gengjie Yang, Timothy Burn, Paul Waeltz, Mark Rupar, Eddy Yue, Brent Douty, Thomas Maduskuie, Nikoo Falahatpisheh, Yun-long Li, Andrew Combs, Gregory Hollis, Reid Huber, Peggy Scherle. The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models. [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 C103.

The role of B-cell receptor inhibitors in the treatment of patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia is a malignancy of mature auto-reactive B cells. Genetic and functional studies implicate B-cell receptor signaling as a pivotal pathway in its pathogenesis. Full B-cell receptor activation requires tumor-microenvironment interactions in lymphoid tissues. Spleen tyrosine kinase, Bruton's tyrosine kinase, and the phosphatidylinositol 3-kinase (PI3K) δ isoform are essential for B-cell receptor signal transduction but also mediate the effect of other pathways engaged in chronic lymphocytic leukemia cells in the tissue-microenvironment. Orally bioavailable inhibitors of spleen tyrosine kinase, Bruton's tyrosine kinase, or PI3Kδ, induce high rates of durable responses. Ibrutinib, a covalent inhibitor of Bruton's tyrosine kinase, and idelalisib, a selective inhibitor of PI3Kδ, have obtained regulatory approval in chronic lymphocytic leukemia. Ibrutinib and idelalisib are active in patients with high-risk features, achieving superior disease control in difficult-to-treat patients than prior best therapy, making them the preferred agents for chronic lymphocytic leukemia with TP53 aberrations and for patients resistant to chemoimmunotherapy. In randomized trials, both ibrutinib, versus ofatumumab, and idelalisib in combination with rituximab, versus placebo with rituximab improved survival in relapsed/refractory chronic lymphocytic leukemia. Responses to B-cell receptor inhibitors are mostly partial, and within clinical trials treatment is continued until progression or occurrence of intolerable side effects. Ibrutinib and idelalisib are, overall, well tolerated; notable adverse events include increased bruising and incidence of atrial fibrillation on ibrutinib and colitis, pneumonitis and transaminase elevations on idelalisib. Randomized trials investigate the role of B-cell receptor inhibitors in first-line therapy and the benefit of combinations. This review discusses the biological basis for targeted therapy of chronic lymphocytic leukemia with B-cell receptor inhibitors, and summarizes the clinical experience with these agents.

Discovery of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (AZD8835): A potent and selective inhibitor of PI3Kα and PI3Kδ for the treatment of cancers.

Starting from potent inhibitors of PI3Kα having poor general kinase selectivity (e.g., 1 and 2), optimisation of this series led to the identification of 25, a potent inhibitor of PI3Kα (wild type, E545K and H1047R mutations) and PI3Kδ, selective versus PI3Kβ and PI3Kγ, with excellent general kinase selectivity. Compound 25 displayed low metabolic turnover and suitable physical properties for oral administration. In vivo, compound 25 showed pharmacodynamic modulation of AKT phosphorylation and near complete inhibition of tumour growth (93% tumour growth inhibition) in a murine H1047R PI3Kα mutated SKOV-3 xenograft tumour model after chronic oral administration at 25mg/kg b.i.d. Compound 25, also known as AZD8835, is currently in phase I clinical trials.

PI3K isoform-selective inhibitors: next-generation targeted cancer therapies.

The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents.

Abstract 4700: The novel and selective PI3Kδ inhibitor, RV1001, displays single agent biologic activity in spontaneous canine NHL

Abstract Background: Non-Hodgkin's lymphoma (NHL) is the most common cancer in dogs with over 70,000 new cases diagnosed each year. Evidence suggests that canine NHL is an excellent spontaneous large animal model of human NHL, particularly for the evaluation of new therapeutics. The purpose of this work was to establish the preclinical profile and clinical efficacy of the novel, potent, and selective PI3Kδ inhibitor RV1001 in canine NHL with ultimate goal of informing the development of such inhibitors in human NHL. Methods: Activity of RV1001 on individual PI3K isoforms was determined using a Homogenous Time Resolved Fluorescence assay (Millipore, Billerica, MA). Potency of the compound on the delta isoform was further corroborated in anti-FcϵR1 induced CD63 expression studies using human whole blood. Ability of RV1001 to arrest growth across several cell lines was determined by a MTT assay. Inhibition of endogenous pAKT in cell lines and primary canine NHL cells was determined by Western blotting. A phase I study of RV1001 was performed in dogs with T and B naïve and drug resistant NHL to assess safety, pharmacokinetic profile and response to therapy. Results: RV1001 demonstrated significant potency against PI3Kδ (39.3 nM) with several fold selectivity over the α (>250), β (>30), and γ (>20) isoforms. In addition, RV1001 inhibited anti-FcϵR1 induced CD63 expression, a marker of PI3Kδ signaling, in human whole blood basophils with an EC50 of 94.7 nM. Proliferation assays in leukemic cells indicated that the compound arrested growth at concentrations between 1.5 and 13 μM. Reduced PI3Kδ signaling was manifested by a reduction in pAKT with half-maximal inhibition noticed between 10 and 100 nM. Oral administration of 100 mg/kg RV1001 to healthy dogs over a 14-day period resulted in significant accumulation (Cmax = 70 μM on Day 1 versus 186 μM on Day 14; PI3Kδ = 69 μM on Day 14). To date, 9 dogs have been entered into the Phase I clinical trial beginning at a dose of 10 mg/kg SID using a standard 3×3 design. One dog with T cell NHL has had a durable complete response to therapy (10 mg/kg), and 3 additional dogs have had partial responses (15 mg/kg n = 2 and 25 mg/kg n = 1) for an objective response rate of 44%. Adverse events associated with RV1001 administration include grade 1 anorexia and grade 3 and 4 LFT elevation. Pharmacokinetic analyses revealed Cmax concentrations of 10-25 μM with AUC0-t ranging from 50 to 100 μM.hr with accumulation evident after 3-4 weeks of dosing. Exploration of alternative dosing regimens is ongoing to eliminate the drug accumulation and thus circumvent the observed hepatotoxicity. Conclusions: RV1001 exhibits potent activity against PI3Kδ with associated inhibition of AKT phosphorylation, excellent oral bioavailability, and strong single agent efficacy in dogs with both B and T NHL. Data generated from these studies will have broad applicability to the further development of isoform specific PI3K inhibitors in people with NHL. Citation Format: Cheryl A. London, Sarah B. Rippy, Misty D. Bear, Kim Cronin, Andrew H. Abbo, Kumar V. Penmetsa, Srikant Viswanadha, Swaroop Vakkalanka. The novel and selective PI3Kδ inhibitor, RV1001, displays single agent biologic activity in spontaneous canine NHL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4700. doi:10.1158/1538-7445.AM2015-4700

Abstract 2665: High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers

Abstract The PIK3CA gene, encoding the p110 catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes described in human cancer. Hence PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. To date most studies with PI3K inhibitors have used a continuous (daily) dosing schedule and although clinical responses have been reported the overall activity observed has been moderate. This may in part be due to suboptimal pathway inhibition which is capped by normal tissue toxicities such as rash, diarrhoea and hyperglycaemia. Furthermore, additional dose reduction may be required when such agents are used in combination with other therapies. Therefore in our efforts to optimise inhibition of PI3K pathway signalling, we have explored high dose intermittent scheduling as an alternative to continuous dosing. Here we describe pre-clinical studies that exemplify such concepts, centred around use of AZD8835, a PI3K inhibitor currently in Phase 1 clinical evaluation. AZD8835 is a novel and potent inhibitor of PI3Kα and PI3Kδ, with selectivity vs. PI3Kβ, PI3Kγ(IC50s of 6nM, 6nM, 431nM and 90nM respectively in enzyme assays) and other kinases. AZD8835 preferentially displays activity in tumour models with a mutant PIK3CA background, such as ER+ve breast cancer models. Such models were used in our investigations, in both cell culture and in mouse xenograft contexts. We demonstrate that a high dose intermittent schedule of single agent AZD8835 achieves greater pathway inhibition yielding significant anti-tumour responses. In the sensitive BT474 xenograft model, a dose of 100mg/kg AZD8835 BID on days 1 and 4 in a weekly schedule delivered -36% tumour regression, accompanied by a strongly elevated rapid onset apoptosis signal with 4-16% cells staining positively for cleaved-caspase3. We also evaluated AZD8835 in combination with other targeted therapeutic agents, in MCF7, BT474 and T47D breast models, observing increased sensitivity relative to single agent AZD8835; firstly with agents that target other nodes in the PI3K pathway; secondly with agents targeting parallel but interconnected driver pathways in breast disease (ER, CDK4/6). Overall the data indicate that high dose intermittent scheduling can deliver strong anti-tumour efficacy in a range of combination settings and provides a promising alternative to continuous dosing. Such schedules merit clinical evaluation. Citation Format: Kevin Hudson, Urs Hancox, Cath Trigwell, Phillippa Dudley, Lyndsey Hanson, Robert McEwen, Alys Jones, Marie Cumberbatch, Urszula Polanska, Rebecca Ellston, Oona Delpuech, Pablo Morentin Gutierrez, Lara Ward, Francisco Cruzalegui, Stephen Green. High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2015-2665

Abstract 2830: Discovery of AZD8835, a potent and selective inhibitor of PI3Kα and PI3Kδ for the treatment of PIK3CA-dependent cancers

Abstract Amplification and mutations of the PIK3CA gene encoding the p110α catalytic unit of PI3Kα occur frequently in many human cancers including ca. 25% of breast cancers. The three most common mutations (E542K, E545K and H1047R) in the PIK3CA gene have been confirmed as activating mutations. The strong evidence pointing at the oncogenic nature of the PIK3CA gene mutations and their high frequency have fuelled a strong interest in developing selective inhibitors of PI3Kα. During the lead generation phase of this project, we identified a series of 2-amino pyridines / pyrazines as potent inhibitors of PI3Kα both at the enzymatic and the cellular level. However, these initial leads displayed poor general kinase selectivity. We report the optimisation of this series, with a special focus on optimisation of general kinase selectivity. This work led to the identification of AZD8835, a potent inhibitor of PI3Kα (wild type, E545K and H1047R mutations) and PI3Kδ with excellent selectivity vs. PI3Kβ, PI3Kγ and an excellent general kinase selectivity. AZD8835 is a potent inhibitor of p-Akt in cells sensitive to PI3Kα inhibition (IC50 0.057 μM in PIK3CA mutant human breast ductal carcinoma BT474 cell line) and in cells sensitive to PI3Kδ inhibition (IC50 0.049 μM in JeKo-1 B cell line), but not to cells sensitive to PI3Kβ inhibition (IC50 3.5 μM in PTEN null breast adenocarcinoma MDA-MB-468 cell line) or PI3Kγ inhibition (IC50 0.53 μM in monocytic RAW264 cell line). Furthermore, AZD8835 displayed high metabolic stability and suitable physical properties for oral administration. In vivo, inhibition of p-Akt was observed at 30 minutes and 8 hours in the PIK3CA H1047R mutant SKOV3 tumour model in mice after chronic oral administration of AZD8835 (25 mg/kg b.i.d.). AZD8835 showed excellent tumour growth inhibition in this same model after chronic oral administration (25 mg/kg b.i.d.). Based on these results, AZD8835 was selected as a clinical candidate for the treatment of PIK3CA-dependant cancers and has recently entered phase I clinical trials. Citation Format: Bernard Barlaam, Sabina Cosulich, Benedicte Delouvrie, Martina Fitzek, Herve Germain, Stephen Green, Craig S. Harris, Kevin Hudson, Christine Lambert-van der Brempt, Maryannick Lamorlette, Le Griffon Antoine, Remy Morgentin, Gilles Ouvry, Ken Page, Georges Pasquet, Linette Ruston, Twana Saleh, Michel Vautier, Lara Ward. Discovery of AZD8835, a potent and selective inhibitor of PI3Kα and PI3Kδ for the treatment of PIK3CA-dependent cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2830. doi:10.1158/1538-7445.AM2015-2830

Abstract 2671: INCB050465, a novel PI3Kδ inhibitor, synergizes with PIM protein kinase inhibition to cause tumor regression in a model of DLBCL

Abstract Phosphatidylinositol 3-kinases (PI3Ks) belong to a family of lipid signaling kinases that phosphorylate phosphatidylinositol-4,5-bisphosphate (PIP2), giving rise to phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. The delta isoform of PI3K (PI3Kδ) plays an essential role in both B cell development and function, and has now been validated as a therapeutic target in CLL and indolent NHL upon the approval of idelalisib, a selective PI3Kδ inhibitor. However, despite the demonstrated activity of PI3Kδ inhibitors in several subtypes of B cell malignancies, the effectiveness of the approach as a single agent for the treatment of DLBCL, the largest subtype of NHL, has not been demonstrated. We hypothesized that an optimal treatment strategy in this histology would require combined inhibition of PI3Kδ with additional agents that target B cells through distinct signaling pathways. INCB050465 is a novel, small molecule inhibitor of PI3Kδ. In biochemical assays, it potently inhibits PI3Kδ (IC50 = 1 nM at 1 mM ATP) with approximately 20,000-fold selectivity for PI3Kα, PI3Kβ, PI3Kγ and 57 other kinases. In various PI3Kδ functional assays, including B cell proliferation induced by activation of BCR, BAFF-R, IL-4R, CD40 and TLRs as well as T cell differentiation assays, INCB050465 demonstrates potent activity with IC50 values ranging from 0.2 to 2 nM. In addition, INCB050465 blocks the proliferation of several DLBCL and MCL cell lines in vitro (EC50 < 10 nM) and can slow tumor growth in the Pfeiffer model of DLBCL in vivo. In cells, INCB050465 blocks constitutive PI3Kδ signaling, including phosphorylation of Akt and FOXO3a, with potencies similar to those for inhibiting proliferation. More importantly, the ability of INCB050465 to inhibit DLBCL cell proliferation in vitro can be further potentiated by inhibition of PIM (Proviral Integration sites for Moloney virus) protein kinases, which were also found to be important for B cell development and to phosphorylate substrates which overlap with those of the PI3K pathway. The combined inhibition of PI3Kδ by INCB050465 with the potent and selective PIM protein kinase inhibitor, INCB053914, results in profound tumor regression in the Pfeiffer cell model of DLBCL. Taken together, these results provide evidence for cross-talk between the PIM and PI3Kδ pathways, and support a mechanistic rationale for the clinical evaluation of combined PIM and PI3Kd inhibition in B cell malignancies, including DLBCL. Citation Format: Niu Shin, Holly Koblish, Maryanne Covington, Yanlong Li, Kathy Wang, Qian Wang, Patricia Feldman, Leslie Hall, Sybil O'Connor, Xin He, Kamna Katiyar, Yu Li, Eddy W. Yue, Thomas P. Maduskuie, Brent Douty, Song Mei, Yun-Long Li, Chu-Biao Xue, Andrew Combs, Wenqing Yao, Sharon Diamond-Fosbenner, Swamy Yeleswaram, Robert Newton, Kris Vaddi, Reid Huber, Peggy Scherle. INCB050465, a novel PI3Kδ inhibitor, synergizes with PIM protein kinase inhibition to cause tumor regression in a model of DLBCL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2015-2671

Abstract IA19: Targeting PI3K-δ and PI3K-γ in hematological malignancies with duvelisib (IPI-145)

Abstract PI3K-δ and PI3K-γ isoforms are preferentially expressed in leukocytes where they have distinct and mostly non-overlapping roles in immune cell function and important roles in the biology of B-cell neoplasms. Duvelisib (IPI-145), a potent, orally available, small molecule inhibitor of PI3K-δ and PI3K-γ, was designed to investigate the hypothesis that simultaneous inhibition of these two specific isoforms would demonstrate broad adaptive and innate immune cell inhibitory activity and enhanced efficacy in hematologic malignancies as well as inflammatory and autoimmune diseases. Biochemical, target occupancy, cellular, and whole blood assays indicate that the inhibition of both the PI3K-δ and PI3K-γ isoforms with duvelisib can be accomplished effectively at clinically relevant concentrations. Duvelisib displays single-agent activity across a spectrum of hematologic neoplasm cell lines and potently inhibits the in vitro proliferation of primary human CLL cells. In certain tumor cell lines, the combination of isoform selective PI3K-δ and PI3K-γ inhibitors demonstrated synergy, suggesting that combined inhibition of both PI3K-δ and PI3K-γ results in greater growth suppression than the inhibition of either isoform alone. In addition, a high throughput combination screen demonstrates synergy between duvelisib in combination with multiple classes of approved and investigational compounds in hematologic malignancies represented by a broad panel of tumor cell lines. Duvelisib is currently in late-stage development for the treatment of hematologic malignancies including iNHL and CLL. Inhibition of both the PI3K-δ and PI3K-γ isoforms targets the malignant cells as well as their microenvironment, suggesting a rationale for the favorable response rates reported in iNHL and CLL patients administered duvelisib. Citation Format: Julian Adams. Targeting PI3K-δ and PI3K-γ in hematological malignancies with duvelisib (IPI-145). [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA19.

Idelalisib monotherapy and durable responses in patients with relapsed or refractory Waldenstroms Macroglobulinemia (WM).

8532 Background: Idelalisib (Zydelig), a selective oral inhibitor of PI3Kδ, demonstrated considerable anti-tumor activity in patients with relapsed/refractory iNHL in phase 1 (p1; Flinn, 2014), and...

Results of a phase III randomized, controlled study evaluating the efficacy and safety of idelalisib (IDELA) in combination with ofatumumab (OFA) for previously treated chronic lymphocytic leukemia (CLL).

7023 Background: IDELA (Zydelig) is a selective oral PI3Kδ inhibitor approved in combination with rituximab for previously treated pts with CLL. This open-label study (NCT01659021) compared IDELA +...

Outcomes of anticoagulant (AC) or antiplatelet (AP) use in patients (pts) with chronic lymphocytic leukemia (CLL) or indolent non-Hodgkin’s lymphoma (iNHL) in idelalisib (IDELA) trials.

8563 Background: IDELA, a selective oral PI3Kδ inhibitor, is approved for use in relapsed CLL (in combination with rituximab [R]) and iNHL (as monotherapy). Both diseases occur mainly in the elderl...

Phosphoinositide 3-kinase β, phosphoinositide 3-kinase δ, and phosphoinositide 3-kinase γ mediate the anti-inflammatory effects of magnesium sulfate

BackgroundWe previously demonstrated that inhibiting phosphoinositide 3-kinase (PI3K) or activating L-type calcium channels blocked the anti-inflammatory effects of magnesium sulfate (MgSO4). However, the question as which class I PI3K isoform (PI3Kα, PI3Kβ, PI3Kδ, or PI3Kγ) is involved in this regard remains unstudied. The question as whether MgSO4 and L-type calcium channels interact to influence PI3K activation also remains unstudied. We therefore designed this study to test two hypotheses: (1) inhibiting PI3Kα, PI3Kβ, PI3Kδ, or PI3Kγ would block the anti-inflammatory effects of MgSO4 and (2) activating L-type calcium channels would block the effects of MgSO4 on activating PI3K. Materials and methodsPI3K isoform investigation: macrophages (RAW264.7 cells) were treated with endotoxin, endotoxin plus MgSO4, or endotoxin plus MgSO4 plus the selective inhibitor of PI3Kα (PIK-75), PI3Kβ (TGX-221), PI3Kδ (IC-87114), or PI3Kγ (AS-252424). Calcium channel investigation: macrophages were treated with endotoxin, endotoxin plus MgSO4, or endotoxin plus MgSO4 plus the L-type calcium channel activator BAY-K8644. ResultsThe endotoxin plus MgSO4 group presented lower concentrations of inflammatory mediators (macrophage inflammatory protein 2, tumor necrosis factor α, and interleukin 6, lower nuclear concentration of phosphorylated nuclear factor κB, lower cytosolic concentration of phosphorylated inhibitor κBα, and higher concentration of phosphorylated Akt (PI3K activation marker) than the endotoxin group (all P < 0.05). These effects of MgSO4 were significantly reduced by TGX-221, IC-87114, or AS-252424, but not PIK-75. Additionally, BAY-K8644 blocked the effect of MgSO4 on activating PI3K. ConclusionsMgSO4 exerts its anti-inflammatory effects through activating PI3Kβ, PI3Kδ, and PI3Kγ. The underlying mechanism appears to involve inhibition of L-type calcium channels.

Synthesis and SAR study of potent and selective PI3Kδ inhibitors

2,3,4-Substituted quinolines such as (10a) were found to be potent inhibitors of PI3Kδ in both biochemical and cellular assays with good selectivity over three other class I PI3K isoforms. Some of those analogs showed favorable pharmacokinetic properties.▪

Discovery of (R)-8-(1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide (AZD8186): a potent and selective inhibitor of PI3Kβ and PI3Kδ for the treatment of PTEN-deficient cancers.

Several studies have highlighted the dependency of PTEN deficient tumors to PI3Kβ activity and specific inhibition of PI3Kδ has been shown activity against human B-cell cancers. We describe the discovery and optimization of a series of 8-(1-anilino)ethyl)-2-morpholino-4-oxo-4H-chromene-6-carboxamides as PI3Kβ/δ inhibitors, which led to the discovery of the clinical candidate 13, also known as AZD8186. On the basis of the lower lipophilicity of the chromen-4-one core compared to the previously utilized pyrido[1,2-a]pyrimid-4-one core, this series of compounds displayed high metabolic stability and suitable physical properties for oral administration. Compound 13 showed profound pharmacodynamic modulation of p-Akt in PTEN-deficient PC3 prostate tumor bearing mice after oral administration and showed complete inhibition of tumor growth in the mouse PTEN-deficient PC3 prostate tumor xenograft model. 13 was selected as a clinical candidate for treatment of PTEN-deficient cancers and has recently entered phase I clinical trials.

The Dual PI3K/mTOR Inhibitor PF-04691502 Induces Substantial Apoptosis in Chronic Lymphocytic Leukemia Cells in Vitro and Prolongs Survival in the Eµ-TCL1 Mouse Model

Pharmacological inhibition of specific B cell receptor signalling pathways within chronic lymphocytic leukemia (CLL) cells offers the potential for improved therapeutic options with reduced off target toxicity. Idelalisib, the PI3Kδ selective inhibitor, has been approved for CLL and significantly improved overall survival among patients with relapsed CLL in combination with rituximab compared to rituximab alone. In addition to PI3Kδ however, there are three other Class I PI3K isoforms, PI3Kα, PI3Kβ and PI3Kγ, with PI3Kα known to have a role in CLL survival and chemotaxis. In neutrophils, functional redundancy between PI3K isoforms is evident, with inhibition of at least three PI3K isoforms required for maximal apoptosis. Inhibition of mTOR is known to induce cell cycle arrest and apoptosis in CLL cells, however prolonged inhibition of mTOR results in activation of a positive feedback loop resulting in PI3K\\Akt reactivation. To overcome these caveats, pan PI3K inhibition alongside mTOR inhibition may achieve superior cytotoxicity against CLL cells compared to PI3Kδ or mTOR inhibition alone. We therefore sought to investigate the effect of a dual pan PI3K and mTOR inhibitor, PF-04691502, on primary CLL cells and in the Eµ-TCL1 mouse model of CLL.Twenty five primary CLL samples were treated with PF-04691502 and downstream signalling and subsequent apoptosis assessed by immunoblotting and Annexin V/PI staining respectively. In primary CLL cells, PF-04691502 induced 80% apoptosis after 24 hours at 10µM concentration. PF-04691502 had an IC50 value of 1µM as assessed by Annexin V/PI staining, with minimal toxicity to normal human B or T cells and a trend towards more specific killing in unmutated CLL (p=0.09) compared to mutated CLL. PF-04691502 inhibited both soluble and immobilised anti-IgM induced signalling and overcame anti-IgM induced survival signals. PF-04691502 abrogated CXCL12 induced signalling and impaired subsequent CLL migration towards the chemokine in a transwell chemotaxis assay. Finally, PF-04691502 was able to overcome protection from co-culture with stroma inducing significant apoptosis of CLL cells when added continuously and in wash out experiments.To assess the effect of PF-04691502 in vivo, twenty Eµ-TCL1 mice were inoculated with tumour cells and after day 21 dosed once daily with vehicle, 5mg/kg or 10mg/kg PF-04691502 for a further 14 days. Mice were assessed for leukemic cell counts, spleen size and white blood cell count throughout the duration of the experiment. The effect of PF-04691502 on B cell receptor and chemokine receptor induced signalling in ex vivo Eµ-TCL1 cells was assessed by immunoblotting.Near identical results to the CLL data above were observed. Using Eµ-TCL1 cells isolated from the spleen, PF-04691502 was shown to profoundly inhibit anti-IgM and CXCL12 induced signalling and chemotaxis as well as induce substantial apoptosis as measured by Annexin V/PI staining. Therefore, we subsequently assessed the effect of PF-04691502 in vivo using this model. Mice treated with PF-04691502 displayed a transient increase in leukemic cell numbers in the blood persisting for 1-4 days, followed by a reduction to levels significantly below that of the vehicle control. The whole white blood cell count remained stable in the PF-04691502 treated mice during the course of treatment, whilst the vehicle treated mice showed significant increases in tumour load up to 40×106 cells/ml 33 days post inoculation. Spleen sizes increased steadily over time in the control mice, whilst PF-04691502 treatment prevented this. After 33 days, PF-04691502 treated mice had spleen sizes comparable to non tumor-recipient control animals. In addition, CD5+ B220+ Eµ-TCL1 cells were significantly reduced in the bone marrow, spleen and lymph nodes following PF-04691502 treatment compared to control mice (p=0.0198; p< 0.0001; p=0.0151 respectively). These results demonstrate that PF-04691502 induces substantial apoptosis of primary CLL cells in vitro and significantly prolongs survival in an in vivo murine model of CLL. Such data indicate that dual pan PI3K/mTOR inhibitors may prove efficacious in the treatment of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

The PI3K Delta Selective Inhibitor Idelalisib Minimally Interferes with Immune Effector Function and B Cell Depletion Mediated By Obinutuzumab (GA101) and Rituximab

Introduction: Idelalisib is a highly selective oral inhibitor of the phosphoinositide 3-kinase delta (PI3Kδ) that is hyperactive in many B-cell malignancies and is critical for the activation, proliferation, survival and trafficking of B lymphocytes. Idelalisib is approved in the US for the treatment of chronic lymphocytic leukemia (CLL) in combination with rituximab and as monotherapy for patients with relapsed follicular B-cell non-Hodgkin lymphoma and small lymphocytic lymphoma who have received at least two prior systemic therapies. Obinutuzumab (GA101) is a glycoengineered type II, CD20 antibody that induces a high level of direct cell death. As a result of glycoengineering, obinutuzumab has increased affinity for FcγRIII on innate immune effector cells resulting in enhanced induction of ADCC and ADCP. Obinutuzumab has been approved for first line treatment of CLL patients in combination with chlorambucil in the US and Europe and is currently in pivotal clinical trials in indolent NHL and DLBCL. Previous work has shown the covalent BTK inhibitor ibrutinib can interfere with the immune effector function and ultimately in vivo efficacy of rituximab in preclinical models (Kohrt et al., Blood, 2014). As PI3K isoforms also play a role in immune effector cells and FcγR signaling we investigated the impact of PI3Kδ inhibition by the PI3Kδ selective inhibitor idelalisib on the immune effector function of obinutuzumab and rituximab.Experimental methods: The impact of idelalisib on NK cell mediated ADCC induction by obinutuzumab and rituximab was investigated in LDH release assays using WIL2-S, SU-DHL4 and Z138 target cells at plasma protein-binding adjusted clinically relevant concentrations mimicking exposure in patients. As a surrogate for NK cell activation CD16 levels and up-regulation of the degranulation marker CD107a were assessed by FACS. The impact on monocyte-derived macrophage mediated ADCP of WIL2-S cells was measured in a flow cytometry-based phagocytosis assay. Finally, depletion of CD19 positive B cells was determined in whole blood from healthy volunteers in flow cytometry-based whole blood assay.Results: In ADCC assays, no impact of idelalisib on ADCC at saturating concentration of obinutuzumab or rituximab (>1ug/ml) can be detected in LDH release assays with tumor cells targets (N=9 donors for WIL2-S, N>3 donors for SU-DHL-4 and Z138). Idelalisib did not alter obinutuzumab or rituximab ability to kill tumor cells by ADCC at low E:T ratio. Little to no increase of obinutuzumab or rituximab EC50 for LDH release, CD16 down regulation, or degranulation of NK cells could be detected depending on donor effector cells. ADCP assays were conducted with M2c polarized macrophages using WIL2-S as targets. Less than 30% inhibition of ADCP was observed in this assay at idelalisib concentration at protein binding-adjusted clinical Cmax. At idelalisib Cmax (4200 nM) the EC50 of obinutuzumab-mediated B cell depletion in healthy human whole blood was increased 3 to 5 times, whereas at Cmin (760 nM) idelalisib did not significantly influence obinutuzumab EC50 or maximal B cell depletion.Conclusions: PI3Kδ inhibition by idelalisib has minimal impact on the immune effector function of obinutuzumab (GA101) and rituximab as measured in NK cell-mediated ADCC, macrophage-mediated ADCP and whole blood B-cell depletion. DisclosuresHerter:Roche: Employment. Palazzo:Gilead Sciences: Employment. Bacac:Roche: Employment. Grosmaire:Gilead Sciences: Employment. Frey:Gilead Sciences: Employment. Pflanz:Gilead Sciences: Employment. Liu:Gilead Sciences: Employment. Tannheimer:Gilead Sciences: Employment. Umana:Roche: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties. Queva:Gilead Sciences: Employment.

BCR pathway inhibition as therapy for chronic lymphocytic leukemia and lymphoplasmacytic lymphoma.

Chronic lymphocytic leukemia (CLL) and lymphoplasmacytic lymphoma (LPL) are malignancies of mature B cells. In LPL, mutations of the adaptor protein MYD88 (L265P) in the Toll-like receptor pathway have been recognized recently as being a hallmark of the disease and indicate a dependence of the tumor on this pathway. In CLL, functional studies have implicated BCR activation in the tissue microenvironment as a pivotal pathway in the pathogenesis. Bruton's tyrosine kinase (BTK) and the PI3Kδ isoform are essential for BCR signaling and also seem to be required for signal transduction in LPL cells, even if the role of BCR signaling in this disease remains less well defined. Ibrutinib, a covalent inhibitor of BTK approved by the Food and Drug Administration as a second-line treatment for CLL, and idelalisib, a selective inhibitor of PI3Kδ, achieve excellent clinical responses in both diseases irrespective of classic markers indicating high-risk disease. Several additional inhibitors targeting BTK and PI3Kδ, as well as the spleen tyrosine kinase, have entered clinical trials. This review discusses the biologic basis for kinase inhibitors as targeted therapy for CLL and LPL and summarizes the clinical experience with these agents.