Neill A. Giese

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.

Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals.

Targeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101-mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell-activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders.

PDGF-D, a new protease-activated growth factor.

Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-α, whereas PDGF-B activates both PDGFR-α and PDGFR-β. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-β but not PDGFR-α. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-α activation may result from PDGFR-α/β heterodimerization.

CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML)

Up to 30% of acute myelogenous leukemia (AML) patients harbor an activating internal tandem duplication (ITD) within the juxtamembrane domain of the FLT3 receptor, suggesting that it may be a target for kinase inhibitor therapy. For this purpose we have developed CT53518, a potent antagonist that inhibits FLT3, platelet-derived growth factor receptor (PDGFR), and c-Kit (IC(50) approximately 200 nM), while other tyrosine or serine/threonine kinases were not significantly inhibited. In Ba/F3 cells expressing different FLT3-ITD mutants, CT53518 inhibited IL-3-independent cell growth and FLT3-ITD autophosphorylation with an IC(50) of 10-100 nM. In human FLT3-ITD-positive AML cell lines, CT53518 induced apoptosis and inhibited FLT3-ITD phosphorylation, cellular proliferation, and signaling through the MAP kinase and PI3 kinase pathways. Therapeutic efficacy of CT53518 was demonstrated both in a nude mouse model and in a murine bone marrow transplant model of FLT3-ITD-induced disease.

PI3K/p110{delta} is a novel therapeutic target in multiple myeloma.

In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110delta signaling in multiple myeloma (MM). Knockdown of p110delta by small interfering RNA caused significant inhibition of MM cell growth. Similarly, p110delta specific small molecule inhibitor CAL-101 triggered cytotoxicity against LB and INA-6 MM cell lines and patient MM cells, associated with inhibition of Akt phosphorylation. In contrast, CAL-101 did not inhibit survival of normal peripheral blood mononuclear cells. CAL-101 overcame MM cell growth conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cell coculture. Interestingly, inhibition of p110delta potently induced autophagy. The in vivo inhibition of p110delta with IC488743 was evaluated in 2 murine xenograft models of human MM: SCID mice bearing human MM cells subcutaneously and the SCID-hu model, in which human MM cells are injected within a human bone chip implanted subcutaneously in SCID mice. IC488743 significantly inhibited tumor growth and prolonged host survival in both models. Finally, combined CAL-101 with bortezomib induced synergistic cytotoxicity against MM cells. Our studies therefore show that PI3K/p110delta is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 to improve patient outcome in MM.

PI3K/p110δ is a novel therapeutic target in multiple myeloma

In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110delta signaling in multiple myeloma (MM). Knockdown of p110delta by small interfering RNA caused significant inhibition of MM cell growth. Similarly, p110delta specific small molecule inhibitor CAL-101 triggered cytotoxicity against LB and INA-6 MM cell lines and patient MM cells, associated with inhibition of Akt phosphorylation. In contrast, CAL-101 did not inhibit survival of normal peripheral blood mononuclear cells. CAL-101 overcame MM cell growth conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cell coculture. Interestingly, inhibition of p110delta potently induced autophagy. The in vivo inhibition of p110delta with IC488743 was evaluated in 2 murine xenograft models of human MM: SCID mice bearing human MM cells subcutaneously and the SCID-hu model, in which human MM cells are injected within a human bone chip implanted subcutaneously in SCID mice. IC488743 significantly inhibited tumor growth and prolonged host survival in both models. Finally, combined CAL-101 with bortezomib induced synergistic cytotoxicity against MM cells. Our studies therefore show that PI3K/p110delta is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 to improve patient outcome in MM.

The Role of Alpha and Beta Platelet-Derived Growth Factor Receptor in the Vascular Response to Injury in Nonhuman Primates

Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.

Blockade of Platelet-Derived Growth Factor or Its Receptors Transiently Delays but Does Not Prevent Fibrous Cap Formation in ApoE Null Mice

Platelet-derived growth factor (PDGF) is a potent stimulant of smooth muscle cell migration and proliferation in culture. To test the role of PDGF in the accumulation of smooth muscle cells in vivo, we evaluated ApoE -/- mice that develop complex lesions of atherosclerosis. Fetal liver cells from PDGF-B-deficient embryos were used to replace the circulating cells of lethally irradiated ApoE -/- mice. One month after transplant, all monocytes in PDGF-B -/- chimeras are of donor origin (lack PDGF), and no PDGF-BB is detected in circulating platelets, primary sources of PDGF in lesions. Although lesion volumes are comparable in the PDGF-B +/+ and -/- chimeras at 35 weeks, lesions in PDGF-B -/- chimeras contain mostly macrophages, appear less mature, and have a reduced frequency of fibrous cap formation as compared with PDGF-B +/+ chimeras. However, after 45 weeks, smooth muscle cell accumulation in fibrous caps is indistinguishable in the two groups. Comparison of elicited peritoneal macrophages by RNase protection assay shows an altered cytokine and cytokine receptor profile in PDGF-B -/- chimeras. ApoE -/- mice were also treated for up to 50 weeks with a PDGF receptor antagonist that blocks all three PDGF receptor dimers. Blockade of the PDGF receptors similarly delays, but does not prevent, accumulation of smooth muscle and fibrous cap formation. Thus, elimination of PDGF-B from circulating cells or blockade of PDGF receptors does not appear sufficient to prevent smooth muscle accumulation in advanced lesions of atherosclerosis.

Functional Importance of Platelet-derived Growth Factor (PDGF) Receptor Extracellular Immunoglobulin-like Domains IDENTIFICATION OF PDGF BINDING SITE AND NEUTRALIZING MONOCLONAL ANTIBODIES

The biological effects of platelet-derived growth factor (PDGF) are mediated by α- and β-PDGF receptors (PDGFR), which have an intracellular tyrosine kinase domain and an extracellular region comprising five immunoglobulin-like domains (D1–D5). Using deletion mutagenesis we mapped the PDGF binding site in each PDGFR to the D2–D3 region. In the case of α-PDGFR,125I-PDGF AA and 125I-PDGF BB bound to the full-length extracellular domain, D1–D5, and D2–D3 with equal affinity (K d = 0.21–0.42 nm). Identical results were obtained for 125I-PDGF BB binding to β-PDGFR mutants D1–D5 and D2–D3, establishing that D1, D4, and D5 do not contribute to PDGF binding. Monoclonal antibodies (mAb) directed against individual PDGFR Ig-like domains were used to extend these observations. The anti-D1 mAb 1E10E2 and anti-D5 mAb 2D4G10 had no effect on α- or β-PDGFR function, respectively. In contrast, mAb 2H7C5 and 2A1E2 directed against D2 of the α- and β-receptor, respectively, blocked PDGF binding, receptor autophosphorylation and mitogenic signaling with IC50 values of 0.1–3.0 nm. An anti-D4 mAb 1C7D5 blocked β-receptor autophosphorylation and signaling without inhibiting PDGF binding consistent with the observation that D4 is essential for PDGFR dimerization (Omura, T., Heldin, C.-H., and Ostman, A. (1997)J. Biol. Chem. 272, 12676–12682). mAbs identified here act as potent PDGFR antagonists that can be used as research tools and potentially as therapeutic agents for the treatment of diseases involving unwanted PDGFR signaling.

PDGF C Is A Selective α Platelet-Derived Growth Factor Receptor Agonist That Is Highly Expressed in Platelet α Granules and Vascular Smooth Muscle

Objective—The platelet-derived growth factor (PDGF) family consists of four members, PDGF A, PDGF B, and 2 new members, PDGF C and PDGF D, which signal through the &agr; and &bgr; PDGF receptor (PDGFR) tyrosine kinases. This study was performed to determine the receptor specificity and cellular expression profile of PDGF C. Methods and Results—PDGF C growth factor domain (GFD) was shown to preferentially bind and activate &agr; PDGFR and activate &bgr; PDGFR when it is co-expressed with &agr; PDGFR through heterodimer formation. An investigation of PDGF C mRNA and protein expression revealed that during mouse fetal development, PDGF C was expressed in the mesonephric mesenchyme, prefusion skeletal muscle, cardiac myoblasts, and in visceral and vascular smooth muscle, whereas in adult human tissues expression was largely restricted to smooth muscle. Microarray analysis of various cell types showed PDGF C expression in vascular smooth muscle cells, renal mesangial cells, and platelets. PDGF C mRNA expression in platelets was confirmed by real-time polymerase chain reaction, and PDGF C protein was localized in &agr; granules by immuno-gold electron microscopy. Western blot analysis of platelets identified 55-kDa and 80-kDa PDGF C isoforms that were secreted on platelet activation. Conclusions—Taken together, our results demonstrated for the first time to our knowledge that like PDGF A and B, PDGF C is likely to play a role in platelet biology.