Potent PI3Kδ Inhibitor Research Articles

Design of Selective Benzoxazepin PI3Kδ Inhibitors Through Control of Dihedral Angles.

A novel selective benzoxazepin inhibitor of PI3Kδ has been discovered. Beginning from compound 3, an αPI3K inhibitor, we utilized structure-based drug design and computational analysis of dihedral torsion angles to optimize for PI3Kδ isoform potency and isoform selectivity. Further medicinal chemistry optimization of the series led to the identification of 24, a highly potent and selective inhibitor of PI3Kδ.

Abstract 143: Preclinical studies on potential therapeutic combination partners for the potent and selective PI3Kδ inhibitor INCB050465 in DLBCL

Abstract The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and function by mediating the signaling of key receptors on B cells. Increased malignant B cell proliferation and survival has also been associated with aberrant activation of PI3Kδ, making selective inhibition of this isoform an attractive therapeutic approach for the treatment of B cell malignancies. INCB050465 is a potent inhibitor of PI3Kδ, with a >20,000 fold selectivity over other PI3K isoforms. Emerging clinical data indicate that INCB050465 monotherapy is well tolerated and results in promising clinical responses in patients with various lymphoma histologies, including those with DLBCL. We therefore sought to explore rational combination strategies for INCB050465 using mouse xenograft models of ABC-subtype (HBL-1), GCB-subtype (Pfeiffer), and GCB/double-hit (WILL-2) human DLBCL, evaluating standard of care agents such as bendamustine and rituximab, as well as with targeted agents. PIM inhibition is a logical addition to PI3Kδ inhibition as a therapeutic approach as both kinases play a critical role in the AKT signaling pathway, having overlapping substrates. Likewise BET inhibition is a rational addition to PI3Kδ inhibition in “double-hit” DLBCL due to de-regulation of MYC transcriptional activity. In vivo studies performed in the Pfeiffer xenograft model demonstrate that INCB050465 combined with the pan-PIM inhibitor INCB053914 yielded complete tumor regressions. This profound decrease in tumor cell survival was due in part to the significant reduction in pBAD levels resulting from dual PIM and PI3Kδ inhibition. Despite modest single agent activity in vivo, the combination of INCB050465 with BET inhibitors, INCB054329 or INCB057643, resulted in significant anti-tumor efficacy in all of the DLBCL models studied, and caused a marked repression in tumor MYC expression. To study the transcriptional effects of combining PI3Kδ and BET inhibitors in this lymphoma model, WILL-2 xenograft tumors from mice treated with single dose INCB050465, INCB054329, the combination, or vehicle control were analyzed by RNAseq. INCB050465 enhanced the ability of INCB054329 to repress a MYC-driven transcriptional program, and the combination also regulated multiple developmental and inflammatory pathways. Together, these data support the clinical evaluation of the PI3Kδ inhibitor INCB050465 as part of a combination regimen with PIM or BET inhibitors for the treatment of DLBCL. Citation Format: Matthew C. Stubbs, Robert Collins, Leslie Hall, Alla Volgina, Holly Koblish, Sang Hyun Lee, Timothy Burn, Phillip C. Liu, Jin Lu, Eddy Yue, Yun-Long Li, Andrew P. Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Preclinical studies on potential therapeutic combination partners for the potent and selective PI3Kδ inhibitor INCB050465 in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 143. doi:10.1158/1538-7445.AM2017-143

Identification of a Potent, Selective, and Efficacious Phosphatidylinositol 3-Kinase δ (PI3Kδ) Inhibitor for the Treatment of Immunological Disorders

PI3Kδ plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4'-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.

Seletalisib: Characterization of a Novel, Potent, and Selective Inhibitor of PI3Kδ.

Phosphoinositide 3-kinases (PI3K) are key signaling enzymes regulating cellular survival, development, and function. Expression of the PI3Kδ isoform is largely restricted to leukocytes and it plays a key role in immune cell development and function. Seletalisib is a novel small-molecule inhibitor of PI3Kδ that was evaluated in biochemical assays, cellular assays of adaptive and innate immunity, and an in vivo rat model of inflammation. Our findings show that seletalisib is a potent, ATP-competitive, and selective PI3Kδ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N-formyl peptide-stimulated but not phorbol myristate acetate-stimulated superoxide release from human neutrophils, consistent with a PI3Kδ-specific activity. No indications of cytotoxicity were observed in peripheral blood mononuclear cells (PBMCs) or other cell types treated with seletalisib. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. Seletalisib showed dose-dependent inhibition in an in vivo rat model of anti-CD3-antibody-induced interleukin 2 release. Collectively, these data characterize seletalisib as a selective PI3Kδ inhibitor and potential therapeutic candidate for the treatment of B-cell malignancies and autoimmune diseases driven by dysregulated proinflammatory cytokine secretion.

Anti-tumor activity of PI3K-δ inhibitor in hematologic malignant cells: Shedding new light on resistance to Idelalisib

Genetic and laboratory experiments have brought remarkable advances in management of human malignancies, which not only revolutionized the understanding of the disease, but also led to development of novel and effective targeted therapies against specific deregulated pathways. This study aimed to investigate anti-cancer effects of Idelalisib, a potent PI3K-δ inhibitor, in a panel of hematological cell lines. The resulting data showed that Idelalisib decreased cell survival in all the tested cell lines; however, as compared to NB4, viability of other cell lines, irrespective of their molecular characteristics or even the compensatory activation of MEK/ERK pathway, was inhibited at higher concentrations. This study suggests for the first time that there is a significant correlation between relative response to Idelalisib and basal expression levels of anti-apoptotic genes, in particular survivin and MCL-1. Intriguingly, we found that Idelalisib-induced apoptosis in NB4, as the most sensitive cell line with the lowest expression level of the aforementioned genes, is executed probably via alteration in the transcriptional level of apoptosis-related genes coupled with p21-mediated caspase-3 activation. Moreover, the lower concentrations of Idelalisib combined with arsenic trioxide (ATO) produced synergistic anti-cancer effect in APL-derived NB4 cells. Overall, due to the pharmacologic safety of Idelalisib and its broad clinical effectiveness in chronic lymphoproliferative disorders, our study suggests that this inhibitor is a promising agent for the treatment of acute promyelocytic leukemia, either as single agent or in a combined-modality strategy.

Identification of highly potent and selective PI3Kδ inhibitors

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.

Discovery of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent and selective PI3Kδ inhibitors

As demonstrated in preclinical animal models, the disruption of PI3Kδ expression or its activity leads to a decrease in inflammatory and immune responses. Therefore, inhibition of PI3Kδ may provide an alternative treatment for autoimmune diseases, such as RA, SLE, and respiratory ailments. Herein, we disclose the identification of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent, selective and orally bioavailable PI3Kδ inhibitors. The lead compound demonstrated efficacy in an in vivo mouse KLH model.

Discovery of a Potent, Selective, and Orally Available PI3Kδ Inhibitor for the Treatment of Inflammatory Diseases.

The delta isoform of the phosphatidylinositol 3-kinase (PI3Kδ) has been shown to have an essential role in specific immune cell functions and thus represents a potential therapeutic target for autoimmune and inflammatory diseases. Herein, the optimization of a series of pyrrolotriazinones as potent and selective PI3Kδ inhibitors is described. The main challenge of the optimization process was to identify an orally available compound with a good pharmacokinetic profile in preclinical species that predicted a suitable dosing regimen in humans. Structure-activity relationships and structure-property relationships are discussed. This medicinal chemistry exercise led to the identification of LAS191954 as a candidate for clinical development.

Discovery of triazole aminopyrazines as a highly potent and selective series of PI3Kδ inhibitors

A novel class of potent PI3Kδ inhibitors with >1000-fold selectivity against other class I PI3K isoforms is described. Optimization of the substituents on a triazole aminopyrazine scaffold, emerging from an in-house PI3Kα program, turned moderately selective PI3Kδ compounds into highly potent and selective PI3Kδ inhibitors. These efforts resulted in a series of aminopyrazines with PI3Kδ IC50⩽1nM in the enzyme assay, some of the most selective PI3Kδ inhibitors published to date, with a cell potency in a JeKo-cell assay of 20–120nM.

Development of single and mixed isoform selectivity PI3Kδ inhibitors by targeting Asn836 of PI3Kδ

A series of PI3Kδ inhibitors derived from the pan-PI3K inhibitor ZSTK474 was prepared that target a non-conserved region of the catalytic site. Dependent upon the substituents present, these analogues show different levels of isoform selectivity and sensitivity to the mutation N836D in PI3Kδ. As a marker of ‘on-target’ activity and permeability, a selection of the most potent PI3Kδ inhibitors were shown to inhibit pAkt production in the Nawalma Burkitt lymphoma cell line.

Discovery and SAR of pyrrolo[2,1-f][1,2,4]triazin-4-amines as potent and selective PI3Kδ inhibitors

Aberrant Class I PI3K signaling is a key factor contributing to many immunological disorders and cancers. We have identified 4-amino pyrrolotriazine as a novel chemotype that selectively inhibits PI3Kδ signaling despite not binding to the specificity pocket of PI3Kδ isoform. Structure activity relationship (SAR) led to the identification of compound 30 that demonstrated efficacy in mouse Keyhole Limpet Hemocyanin (KLH) and collagen induced arthritis (CIA) models.

Abstract CT056: Preliminary safety, efficacy, and pharmacodynamics of a highly selective PI3Kδ inhibitor, INCB050465, in patients with previously treated B-cell malignancies

Abstract Background: PI3Kδ inhibitors have demonstrated efficacy in the treatment of B-cell malignancies, but off-target toxicity and/or the inability to achieve near-complete inhibition of the PI3Kδ pathway may affect the depth and duration of response. INCB050465 is a novel, potent, and selective PI3Kδ inhibitor (Shin et al. AACR 2015. Abstract 2671), with a differentiated profile for potency (whole blood IC50 = 10 nM), dose (<50 mg total daily dose), and preclinical safety (lack of hepatoxicity up to exposures that exceed IC90 coverage by >10-fold). An open-label phase 1 study was initiated to evaluate INCB050465 alone or in combination with other agents in patients with previously treated B-cell malignancies; preliminary results of the dose escalation of INCB050465 monotherapy are reported. Methods: Adult patients with B-cell malignancies who were refractory to or for whom available treatments had failed were eligible. Dose escalation was conducted with a 3 + 3 design after an initial single patient cohort. Patients were observed for 21 days before enrollment of the next cohort. Results: As of the data cutoff, 15 patients were enrolled and treated with INCB050465 at doses ranging from 5 to 30 mg once daily. The median age was 64 years and 47% were men. Patients received a median of 2 prior treatment regimens and the median time since initial diagnosis was 4.9 years. Disease subtypes included DLBCL (n = 6), CLL (n = 3), FL (n = 2), MZL (n = 2), MCL (n = 1), and HL (n = 1). Median exposure to INCB050465 was 15.4 weeks (range, 6.9 to 36.4+ weeks) and no patients required dose reduction. INCB050465 was well tolerated with no DLTs observed. Five patients discontinued treatment, 4 due to disease progression and 1 due to an adverse event (grade 2 exfoliative dermatitis). Ten grade 3 or greater treatment-emergent adverse events were seen in 6 patients: anemia (n = 2), bacteremia, Escherichia infection, hypotension, neutropenia, pneumonia, sepsis, syncope, and WBC count decreased (n = 1 each). To date, only grade 1 transaminase elevations have been observed. Responses were observed in DLBCL (3/6 patients), CLL (1/3), FL (2/2), MZL (2/2), and MCL (1/1). Clinical benefit was observed early in the course of treatment, and the longest duration of response was 27+ weeks. Terminal half-life and suppression of PI3Kδ signaling at trough support once daily dosing. Conclusions: INCB050465 monotherapy was generally well tolerated at all doses tested. Robust and durable signaling pathway inhibition was achieved at all doses and INCB050465 showed promising efficacy in FL, DLBCL, and other lymphomas, with a potentially favorable toxicity profile vs other PI3Kδ inhibitors. Enrollment in dose-escalation, combination therapy, and disease-specific cohorts is ongoing. Citation Format: Andres Forero-Torres, Michael S. Wertheim, Tycel J. Phillips, Martin E. Gutierrez, William J. Edenfield, Swamy Yeleswaram, Maureen Bleam, Li Zhou, Jennifer H. Pulini, Théa Faivre, Harry Miao, Matt Spear, Rod Ramchandren. Preliminary safety, efficacy, and pharmacodynamics of a highly selective PI3Kδ inhibitor, INCB050465, in patients with previously treated B-cell malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT056.

Combination therapy with copanlisib and ABL tyrosine kinase inhibitors against Philadelphia chromosome-positive resistant cells.

ABL tyrosine kinase inhibitor (TKI) therapy has improved the survival of patients with Philadelphia (Ph) chromosome-positive leukemia. However, ABL TKIs cannot eradicate leukemia stem cells. Therefore, new therapeutic approaches for Ph-positive leukemia are needed. Aberrant activation of phosphoinositide 3-kinase (PI3K) signaling is important for the initiation and maintenance of human cancers. Copanlisib (BAY80-6946) is a potent inhibitor of PI3Kα and PI3K-δ. Here we investigated the efficacy of combination therapy of copanlisib with an ABL TKI (imatinib, nilotinib, or ponatinib) using BCR-ABL-positive cells. Although the effects of the ABL TKI treatment were reduced in the presence of the feeder cell line, HS-5, copanlisib inhibited cell growth. Upon combining ABL TKI and copanlisib, cell growth was reduced. Ponatinib and copanlisib combined therapy reduced tumor volume and increased survival in mouse allograft models, respectively. These results indicate that the PI3Kα and -δ inhibitors overcame the chemoprotective effects of the feeder cells and enhanced ABL TKI cytotoxicity. Thus, co-treatment with ABL TKI and copanlisib may be a powerful strategy against ABL TKI-resistant cells, including those harboring the related T315I mutation.

Intermittent High-Dose Scheduling of AZD8835, a Novel Selective Inhibitor of PI3Kα and PI3Kδ, Demonstrates Treatment Strategies for PIK3CA-Dependent Breast Cancers.

The PIK3CA gene, encoding the p110α catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes in human cancer. Hence, PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. Here, we report studies with a novel PI3K inhibitor, AZD8835, currently in phase I clinical evaluation. AZD8835 is a potent inhibitor of PI3Kα and PI3Kδ with selectivity versus PI3Kβ, PI3Kγ, and other kinases that preferentially inhibited growth in cells with mutant PIK3CA status, such as in estrogen receptor-positive (ER(+)) breast cancer cell lines BT474, MCF7, and T47D (sub-μmol/L GI50s). Consistent with this, AZD8835 demonstrated antitumor efficacy in corresponding breast cancer xenograft models when dosed continuously. In addition, an alternative approach of intermittent high-dose scheduling (IHDS) was explored given our observations that higher exposures achieved greater pathway inhibition and induced apoptosis. Indeed, using IHDS, monotherapy AZD8835 was able to induce tumor xenograft regression. Furthermore, AZD8835 IHDS in combination with other targeted therapeutic agents further enhanced antitumor activity (up to 92% regression). Combination partners were prioritized on the basis of our mechanistic insights demonstrating signaling pathway cross-talk, with a focus on targeting interdependent ER and/or CDK4/6 pathways or alternatively a node (mTOR) in the PI3K-pathway, approaches with demonstrated clinical benefit in ER(+) breast cancer patients. In summary, AZD8835 IHDS delivers strong antitumor efficacy in a range of combination settings and provides a promising alternative to continuous dosing to optimize the therapeutic index in patients. Such schedules merit clinical evaluation. Mol Cancer Ther; 15(5); 877-89. ©2016 AACR.

Characterization of selective and potent PI3Kδ inhibitor (PI3KDIN- 015) for B-Cell malignances.

PI3Kδ is predominately expressed in leukocytes and has been found overexpressed in B-cell related malignances such as CLL and AML. We have discovered a highly selective ATP competitive PI3Kd inhibitor PI3KD-IN-015, which exhibits a high selectivity among other PI3K isoforms in both biochemical assays and cellular assay, meanwhile did not inhibit most of other protein kinases in the kinome. PI3KD-IN-015 demonstrates moderately anti-proliferation efficacies against a variety of B-cell related cancer cell lines through down-regulate the PI3K signaling significantly. It induced both apoptosis and autophagy in B-cell malignant cell lines. In addition, combination of autophagy inhibitor Bafilomycin could potentiate the moderate anti-proliferation effect of PI3KD-IN-015. PI3KD-IN-015 shows anti-proliferation efficacy against CLL and AML patient primary cells. Collectively, these results indicate that PI3KD-IN-015 may be useful drug candidate for further development of anti-B-cell related malignances therapies.

Discovery, Optimization, and in Vivo Evaluation of Benzimidazole Derivatives AM-8508 and AM-9635 as Potent and Selective PI3Kδ Inhibitors.

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

Population pharmacokinetic modeling of idelalisib, a novel PI3Kδ inhibitor, in healthy subjects and patients with hematologic malignancies.

Idelalisib is a potent PI3Kδ inhibitor that was recently approved for treating hematologic malignancies. The objective of this analysis was to develop a population pharmacokinetic model for idelalisib and its inactive metabolite GS-563117 and to evaluate the impact of covariates on idelalisib/GS-563117 PK. Data from 10 phase I or II studies in healthy volunteers or patients with hematologic malignancies (n = 736) were analyzed using NONMEM. Stepwise forward addition followed by backward elimination was implemented in the covariate (age, gender, race, body weight, baseline CLcr, AST, ALT, disease status, and type of cancer) model building process. Various model assessment methods were used to evaluate the models. Idelalisib plasma PK was best described by a two-compartment model with first-order absorption, first-order elimination from the central compartment, and a lag time. A nonlinear relationship between dose and relative bioavailability was included in the final model. Two statistically significant covariates were identified and incorporated into the final model: health status (healthy vs. patient) on CL/F and Q/F and body weight on CL/F. Despite being a statistically significant covariate, the effect of body weight on idelalisib exposures was weak, as evidenced by minor changes of steady-state exposure (C trough: 16%; AUC and C max: 10%) for a patient with extreme body weight (5th and 95th percentile) relative to the typical patient, and not considered to be clinically relevant. PopPK models were developed to adequately describe the plasma concentrations of idelalisib and GS-563117. There were no covariate that had a clinically meaningful impact on idelalisib or GS-563117 exposure.

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.

Up-Regulation of the PI3K Signaling Pathway Mediates Resistance to Idelalisib

Background: Idelalisib (Zydelig®), a potent and selective PI3Kδ inhibitor, was recently approved for the treatment of relapsed CLL, SLL and FL. PI3Kδ is critical for malignant B-cell proliferation, survival and homing, and inhibition results in a rapid decrease in lymphadenopathy and clinical response (Yang et al., Clin Can Res 2015). Although idelalisib is very potent in mitigating disease, most patients experience incomplete response and ultimately progress. To understand the mechanism of resistance, we screened ABC-DLBCL cell lines for sensitivity to idelalisib and selected TMD8, an ABC-DLBCL cell line, for the generation of idelalisib resistant cells. We report on the mechanisms of resistance and on downstream biomarkers associated with sensitivity to idelalisib in lymphoma cell lines.Methods: Growth inhibition to idelalisib or other inhibitors was assessed using CellTiterGlo viability assay (Promega) at 96 h. Idelalisib resistant line (TMD8R) was generated by continuous exposure to1μM idelalisib (~2x Cmax corrected for protein binding). A DMSO passage matched line was generated as control (TMD8S). Clonal isolates from pools were generated through 2 rounds of limiting dilution. Cell lines were analyzed by whole exome sequencing (WES, GeneWiz), RNASeq (Expression Analysis) and phosphoproteomics (MultiPathway PTMScan Direct, Cell Signaling Technologies). Protein expression was measured using Simple Western (Protein Simple) and SDS/PAGE Western blot.Results: TMD8 were sensitive to idelalisib and the pan PI3K inhibitor (GDC-0941) with an EC50 of 42 and 27nM, respectively, but not to PI3Kα (BYL-719) or PI3Kβ (AZD-6482) inhibitors, indicating that cell viability is mostly driven by PI3Kδ. TMD8 cells with acquired resistance to idelalisib (TMD8R) showed a loss of sensitivity to idelalisib, with a growth inhibition of 19% vs. 92% at 1μM as compared to TMD8S. No mutation in PIK3CD or other PI3K pathway members, including PTEN, was found in TMD8R clones by WES. In 8/8 TMD8R clones a dramatic reduction of PTEN protein expression was observed (9-fold) despite normal expression of PTEN mRNA by RNAseq (Figure 1A). Additionally 8/8 TMD8R clones showed a modest up-regulation of PIK3CG mRNA (2-fold) and protein (3-5 fold, Figure 1B). PIK3CD remained the most prevalent PI3K isoform expressed in all TMD8R clones (Figure 1C). TMD8R were cross-resistant to the dual PI3Kδ/γ inhibitor duvelisib (EC50 > 4 uM for TMD8R vs. EC50 = 58 nM for TMD8S). RNAseq analysis of idelalisib sensitive and resistant ABC-DLBCL cell lines showed that idelalisib treatment led to c-Myc mRNA down regulation in sensitive cell lines (TMD8 and Ri-1) but not in the resistant cell lines (U2932 and SU-DHL-8). In addition, expression of c-Myc target genes was unchanged in the TMDR. Analysis of phosphoproteins showed PI3K and MAPK pathway up-regulation in TMDR, compared to TMD8S, with increased expression of p-Akt T308, p-Akt S473, p-PDK1 S241, p-GSK3β S9 and p-ERK T202/Y204 (11-, 6-, 2-, 1- and 8-fold, respectively), providing a potential mechanism for the loss of c-Myc down regulation in resistant cells (Wen-Bin Tsai et al., Cancer Res, 2012). Other B-cell receptor signaling proteins (p-BTK Y223, p-SYK Y525/526, and p-STAT-5 Y694) were not detected or unchanged. TMD8R cells were cross resistant to the BTK inhibitors ibrutinib and ONO/GS-4059. Akt (MK-2206) or PDK1 (GSK233440) inhibitors were less potent in TMD8R vs. TMD8S cells, yet in combination with idelalisib at 1μM, their potency was restored to the level observed in TMD8S, indicating that resistant cells retain some dependency on the PI3Kδ.Conclusions: Resistance to idelalisib in the ABC-DLBCL cell line TMD8 was not acquired through de novo mutations. Rather loss of PTEN protein expression, modest up regulation of PI3Kγ, activation of the PI3K and MAPK pathway and loss of c-Myc down regulation by idelalisib were identified as potential mechanism of resistance. Treatment with agents targeting the PI3K pathway in combination with idelalisib may overcome resistance. [Display omitted] DisclosuresMeadows:Gilead Sciences: Employment, Other: Share holder. Rick:Gilead Sciences: Employment, Other: stock holder. Anella:Gilead Sciences: Employment, Other: Stock holder. Liu:Gilead Sciences: Employment, Other: stock holder. Li:Gilead Sciences: Employment, Other: Share holder. Yue:Gilead Sciences: Employment, Other: Share holder. Queva:Gilead Sciences: Other: Stock holder. Tannheimer:Gilead Sciences: Employment, Other: Share holder.

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.