Bart H. Steiner

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.

Structural and Functional Definition of the Human Chitinase Chitin-binding Domain

Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has been detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected withAspergillus. Its normal physiological function is unknown. To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain. The C-terminal 49 amino acids make up the minimal sequence required for chitin binding activity. The absence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of insoluble chitin. Within the minimal chitin-binding domain are six cysteines; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain revealed the presence of three disulfide linkages. The recombinant domain binds specifically to chitin but does not bind chitosan, cellulose, xylan, β-1,3-glucan, β-1,3–1,4-glucan, or mannan. Fluorescently tagged chitin-binding domain was used to demonstrate chitin-specific binding toSaccharomyces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa. These experiments define structural features of the minimal domain of human chitinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal cell wall.

Macrophage-Derived Chemokine and EBI1-Ligand Chemokine Attract Human Thymocytes in Different Stage of Development and Are Produced by Distinct Subsets of Medullary Epithelial Cells: Possible Implications for Negative Selection

The chemoattractant activity of macrophage-derived chemokine (MDC), EBI1-ligand chemokine (ELC), and secondary lymphoid tissue chemokine (SLC) on human thymocytes was analyzed. Both ELC and SLC caused the accumulation of CD4+CD8− or CD4−CD8+ CD45RA+ thymocytes showing high CD3 expression. By contrast, a remarkable proportion of MDC-responsive thymocytes were CD4+CD8+ cells exhibiting reduced levels of CD8 or CD4+CD8− cells showing CD3 and CD45R0, but not CD45RA. MDC-responsive thymocyte suspensions were enriched in cells expressing the MDC receptor, CCR4, selectively localized to the medulla, and in CD30+ cells, whereas ELC-responsive thymocytes never expressed CD30. Reactivity to both MDC and ELC was localized to cells of the medullary areas, but never in the cortex. Double immunostaining showed no reactivity for either MDC or ELC by T cells, macrophages, or mature dendritic cells, whereas many medullary epithelial cells were reactive to MDC or ELC. However, MDC reactivity was consistently localized to the outer wall of Hassal’s corpuscles, whereas ELC reactivity was often found in cells surrounding medullary vessels, but not in Hassal’s corpuscles. Moreover, while most MDC-producing cells also stained positive for CD30L, this molecule was never found on ELC-producing cells. We suggest therefore that CD30L-expressing MDC-producing medullary epithelial cells attract CCR4-expressing thymocytes, thus favoring the CD30/CD30L interaction, and therefore the apoptosis, of cells that are induced to express CD30 by autoantigen activation. By contrast, ELC production by CD30L-lacking medullary epithelial cells may induce the migration into periphery of mature thymocytes that have survived the process of negative selection.

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.

Inhibition of PI3Kδ Reduces Kidney Infiltration by Macrophages and Ameliorates Systemic Lupus in the Mouse

Systemic lupus erythematosus (SLE) is a human chronic inflammatory disease generated and maintained throughout life by autoreactive T and B cells. Class I phosphoinositide 3-kinases (PI3K) are heterodimers composed of a regulatory and a catalytic subunit that catalyze phosphoinositide-3,4,5-P3 formation and regulate cell survival, migration, and division. Activity of the PI3Kδ isoform is enhanced in human SLE patient PBLs. In this study, we analyzed the effect of inhibiting PI3Kδ in MRL/lpr mice, a model of human SLE. We found that PI3Kδ inhibition ameliorated lupus progression. Treatment of these mice with a PI3Kδ inhibitor reduced the excessive numbers of CD4+ effector/memory cells and B cells. In addition, this treatment reduced serum TNF-α levels and the number of macrophages infiltrating the kidney. Expression of inactive PI3Kδ, but not deletion of the other hematopoietic isoform PI3Kγ, reduced the ability of macrophages to cross the basement membrane, a process required to infiltrate the kidney, explaining MRL/lpr mice improvement by pharmacologic inhibition of PI3Kδ. The observations that p110δ inhibitor prolonged mouse life span, reduced disease symptoms, and showed no obvious secondary effects indicates that PI3Kδ is a promising target for SLE.

2,4,6-Triaminopyrimidine as a Novel Hinge Binder in a Series of PI3Kδ Selective Inhibitors

Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) is an appealing target for several hematological malignancies and inflammatory diseases. Herein, we describe the discovery and optimization of a series of propeller shaped PI3Kδ inhibitors comprising a novel triaminopyrimidine hinge binder. Combinations of electronic and structural strategies were employed to mitigate aldehyde oxidase mediated metabolism. This medicinal chemistry effort culminated in the identification of 52, a potent and highly selective inhibitor of PI3Kδ that demonstrates efficacy in a rat model of arthritis.

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.

Dose-Dependent Suppression of Cytokine production from T cells by a Novel Phosphoinositide 3-Kinase Delta Inhibitor.

There remains a significant need for development of effective small molecules that can inhibit cytokine-mediated inflammation. Phosphoinositide 3 kinase (PI3K) is a direct upstream activator of AKT, and plays a critical role in multiple cell signaling pathways, cell cycle progression, and cell growth, and PI3K inhibitors have been approved or are in clinical development. We examined novel PI3Kdelta inhibitors, which are highly selective for the p110delta isoform of in CD3/CD28 stimulated T-cell cytokine production. In vitro generated CD4+ T effector cells stimulated in the presence of a PI3Kdelta inhibitor demonstrated a dose-dependent suppression of cytokines produced by Th1, Th2, and Th17 cells. This effect was T-cell intrinsic, and we observed similar effects on human PBMCs. Th17 cells expressing a constitutively activated form of AKT were resistant to PI3Kdelta inhibition, suggesting that the inhibitor is acting through AKT signaling pathways. Additionally, PI3Kdelta inhibition decreased IL-17 production in vivo and decreased neutrophil recruitment to the lung in a murine model of acute pulmonary inflammation. These experiments show that targeting PI3Kdelta activity can modulate T-cell cytokine production and reduce inflammation in vivo, suggesting that PI3Kdelta inhibition could have therapeutic potential in treating inflammatory diseases.

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.