Adam Kashishian

CAL-101, a p110δ selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability

Phosphatidylinositol-3-kinase p110δ serves as a central integration point for signaling from cell surface receptors known to promote malignant B-cell proliferation and survival. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ as a treatment approach for patients with B-cell malignancies. We thus identified 5-fluoro-3-phenyl-2-[(S)-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one (CAL-101), a highly selective and potent p110δ small molecule inhibitor (half-maximal effective concentration [EC(50)] = 8nM). Using tumor cell lines and primary patient samples representing multiple B-cell malignancies, we have demonstrated that constitutive phosphatidylinositol-3-kinase pathway activation is p110δ-dependent. CAL-101 blocked constitutive phosphatidylinositol-3-kinase signaling, resulting in decreased phosphorylation of Akt and other downstream effectors, an increase in poly(ADP-ribose) polymerase and caspase cleavage and an induction of apoptosis. These effects have been observed across a broad range of immature and mature B-cell malignancies, thereby providing a rationale for the ongoing clinical evaluation of CAL-101.

PI3Kδ inhibitor, GS-1101(CAL-101), attenuates pathway signaling, induces apoptosis, and overcomes signals from the microenvironment in cellular models of Hodgkin lymphoma

GS-1101 (CAL-101) is an oral PI3Kδ-specific inhibitor that has shown preclinical and clinical activity in non-Hodgkin lymphoma and chronic lymphocytic leukemia. To investigate the potential role of PI3Kδ in Hodgkin lymphoma (HL), we screened 5 HL cell lines and primary samples from patients with HL for PI3Kδ isoform expression and constitutive PI3K pathway activation. Inhibition of PI3Kδ by GS-1101 resulted in the inhibition of Akt phosphorylation. Cocultures with stroma cells induced Akt activation in HL cells, and this effect was blocked by GS-1101. Conversely, production of the stroma-stimulating chemokine, CCL5, by HL cells was reduced by GS-1101. GS-1101 also induced dose-dependent apoptosis of HL cells at 48 hours. Reductions in cell viability and apoptosis were enhanced when combining GS-1101 with the mTOR inhibitor everolimus. Our findings suggest that excessive PI3Kδ activity is characteristic in HL and support clinical evaluation of GS-1101, alone and in combination, as targeted therapy for HL.

Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts ACTIONS ON CYTOSKELETAL ORGANIZATION, SURVIVAL, AND RESORPTION

Background: Little is known about the function of specific phosphatidylinositol 3-kinase (PI3K) isoforms in osteoclasts. Results: Using a panel of isoform-selective inhibitors, we found that PI3Kδ regulates osteoclast morphology, actin cytoskeletal organization, and resorptive activity. Conclusion: The PI3Kδ isoform plays a critical role in regulating osteoclast resorptive activity. Significance: PI3Kδ is an attractive target for anti-resorptive therapeutics. Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.

Discovery of Orally Efficacious Phosphoinositide 3-Kinase δ Inhibitors with Improved Metabolic Stability

Aberrant signaling of phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in numerous pathologies including hematological malignancies and rheumatoid arthritis. Described in this manuscript are the discovery, optimization, and in vivo evaluation of a novel series of pyridine-containing PI3Kδ inhibitors. This work led to the discovery of 35, a highly selective inhibitor of PI3Kδ which displays an excellent pharmacokinetic profile and is efficacious in a rodent model of rheumatoid arthritis.

Abstract 2673: Idelalisib has activity at clinically achievable drug concentrations in a subset of ABC and GCB diffuse large B-cell lymphoma and transformed follicular lymphoma cell lines

Diffuse large B-cell lymphoma (DLBCL) is the largest lymphoma subtype representing approximately one-third of all cases of non-Hodgkin9s lymphoma. Gene expression profiling showed that DLBCL can be stratified into activated B-cell (ABC) or germinal center B-cell (GCB) subtypes (Alizadeh et al., Nature 2000). The overall five year survival rate is only approximately 50% (Shaffer et al., Annu. Rev. Immunol. 2012), thus a medical need exits for the treatment of this disease, particularly for patients who relapse after the first line chemoimmunotherapy regimen. Idelalisib is an investigational, highly selective oral inhibitor of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) delta isoform. PI3K delta signaling is required for B lymphocyte activation, proliferation and survival and is dysregulated in several B-cell malignancies. We investigated the activity of idelalisib and ibrutinib, an inhibitor of Bruton9s tyrosine kinase, on the inhibition of proliferation against a panel of 22 DLBCL (9 ABC, 13 GCB) and 3 transformed follicular lymphoma cell lines. Idelalisib has potent activity (EC50 Citation Format: Jia Y. Liu, Tom Kenney, Leslie Butterworth, Adam Kashishian, Sarah Meadows, Peng Yue, Li Li, Kathleen Keegan, Christophe Queva, Stacey Tannheimer. Idelalisib has activity at clinically achievable drug concentrations in a subset of ABC and GCB diffuse large B-cell lymphoma and transformed follicular lymphoma cell lines. [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 2673. doi:10.1158/1538-7445.AM2015-2673

Discovery of a Phosphoinositide 3-Kinase (PI3K) β/δ Inhibitor for the Treatment of Phosphatase and Tensin Homolog (PTEN) Deficient Tumors: Building PI3Kβ Potency in a PI3Kδ-Selective Template by Targeting Nonconserved Asp856

Phosphoinositide 3-kinase (PI3K) β signaling is required to sustain cancer cell growth in which the tumor suppressor phosphatase and tensin homolog (PTEN) has been deactivated. This manuscript describes the discovery, optimization, and in vivo evaluation of a novel series of PI3Kβ/δ inhibitors in which PI3Kβ potency was built in a PI3Kδ-selective template. This work led to the discovery of a highly selective PI3Kβ/δ inhibitor displaying excellent pharmacokinetic profile and efficacy in a human PTEN-deficient LNCaP prostate carcinoma xenograft tumor model.

Abstract 3555: Anti-tumor activity of CAL-101, a potent selective inhibitor of the p110Δ isoform of PI3K, in models of human glioblastoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation play a pivotal role in cell proliferation and survival, transducing signals from several cell surface receptors through PI3K isoforms (designated p110α, β, Δ, and γ). Our previous research has elucidated a significant role of the PI3K p110Δ (PI3KΔ) isoform in hematologic malignancies. Aberrant upregulation of the PI3K/Akt pathway is a frequently observed defect in human malignancies, including glioblastoma, but the role of the PI3KΔ isoform, has not been systematically evaluated. Selective inhibition of PI3KΔ with the orally bioavailable investigational drug, CAL-101, offers the potential for targeting PI3KΔ as a therapeutic approach to glioblastoma while avoiding undesirable side effects associated with pan-inhibitors that broadly block PI3K isoforms in multiple cell types. CAL-101 potently inhibits PI3KΔ (with an EC50 of 65 nM in a whole-blood assay) and shows >200-fold selectivity over other Class I PI3K isoforms and no activity against Class II and III PI3K family members or other PI3K-related proteins, including mTOR and DNA-PK. We examined expression levels of PI3KΔ and constitutive pathway activation in a panel of glioma cell lines (LN18, LN229, SF-539, U87MG, U138MG, and U251). PI3KΔ was consistently expressed and was functionally active, inducing high basal levels of phosphorylated-Akt (pAkt) in all tested cell lines. Treatment with CAL-101 decreased p-Akt levels in all cell lines and also reduced phosphorylation of the downstream pathway target, S6. Furthermore, PI3KΔ inhibition with CAL-101 resulted in an accumulation of cells in G1 and a decrease in the S phase population. Consistent with the effects of CAL-101 on G1 cell-cycle arrest, immunoblotting showed a decrease in cyclin D1 levels. In mice bearing U87MG xenografts, in vivo PI3KΔ inhibition resulted in significant anti-tumor effects, delaying time to tumor progression (tumor volume of >1000 mm3) by 30 days (p<0.001). Taken together, these data suggest that PI3KΔ overexpression and heightened functional activity contribute to glioblastoma proliferation, and that selective targeting of PI3KΔ may offer a novel therapeutic approach for patients with central nervous system tumors. 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 3555. doi:10.1158/1538-7445.AM2011-3555

Abstract B136: CAL‐120, a novel dual p110β/p110δ phosphatidylinositol‐3‐kinase (PI3K) inhibitor, attenuates PI3K signaling and demonstrates potent in vivo antitumor activity against solid tumors

Phosphatidylinositide 3‐kinases (PI3K) are a family of lipid kinases that are involved in signaling events which control a diverse number of cellular processes. The activation of the PI3K pathway by cell surface receptors is directly mediated by the class I isoforms (α, β, δ, and γ). Aberrant regulation of the PI3K signaling pathway is frequently observed in a wide range of human malignancies including gain‐of‐function mutations in PI3K p110α isoform and/or loss‐of‐function mutations in PTEN phosphatase, which is responsible for down regulation of PI3K signaling. In purified enzyme assays, CAL‐120, a dual p110β/p110δ PI3K inhibitor, was inactive against class II and III PI3K family members,the PI3K‐related protein kinases mTOR and DNA‐PK as well as an additional ∼350 protein kinases in a genome wide screen. The ability of CAL‐120 to block oncogenic transformation mediated by individual PI3K class Ia isoforms was evaluated in primary cells using viral transduction. Foci formation mediated by p110α and p110δ was inhibited at 15–200 nM whereas little or no inhibition was observed against oncogenic forms of p110α at 20‐fold higher concentrations. To further demonstrate p110 isoform selectivity, AKT phosphorylation was induced in embryonic fibroblasts with PDGF or LPA that is mediated by p110α and p110β respectively. CAL‐120 inhibited the p110β response with an IC50 of 1.2 µM whereas the p110α IC50 was greater than 20 µM. The antitumor activity of CAL‐120 was evaluated in a panel of 23 human tumor cell lines representing different tissues and PI3K pathway mutations. Constitutive PI3K pathway activation as measured by AKT phosphorylation was observed in 50% of the cell lines and was highly correlated with PTEN mutations. In all cases, CAL‐120 blocked AKT phosphorylation at concentrations of 0.1–1.0 µM. In most cases inhibition of the phosphorylation of downstream effectors Akt, GSK‐3 , and S6 ribosomal protein was also observed over this concentration range. These effects of CAL‐120 on PI3K pathway inhibition correlated with G1 cell cycle arrest leading to inhibition of tumor cell proliferation and in a number of cases induced apoptosis. Of note was a lack of PI3K pathway activation in cell lines with K‐RAS mutations and their insensitivity to CAL‐120 treatment. In mice bearing xenografts of MCF‐7 breast adenocarcinoma (p110α mutation), PC‐3 prostate adenocarcinoma (PTEN deficient), or OVCAR‐3 ovarian adenocarcinoma (no pathway mutation), oral administration of CAL‐120 significantly inhibited tumor growth or caused tumor regression in each of these models. These data are the first to demonstrate that p110β/δ inhibition in the absence of effects on p110α is an effective strategy for the treatment of solid tumors. The antitumor activity was not restricted to cells with PTEN loss and was surprisingly observed even when p110α mutations were present. Collectively, these preclinical data support clinical evaluation of CAL‐120, an oral dual p110α/p110δ inhibitor, for the treatment of patients with solid tumors. (L. U. and P.K.V. are supported by grants from the National Cancer Institute. This is manuscript number 20362 of The Scripps Research Institute). Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B136.