ALBERT

Description: The mammalian target of rapamycin (mTOR) is an intracellular protein that acts as a central regulator of multiple signaling pathways (IGF, EGF, PDGF, VEGF, amino acids) that mediate abnormal growth, proliferation, survival and angiogenesis in cancer. mTOR is a critical component of the PI3K/Akt pathway, a key cell survival pathway that is dysregulated in many cancers including multiple myeloma (MM). mTOR is an important therapeutic target because it is a “rate-limiting” bottleneck in the key signaling pathway that regulates cell survival, proliferation, and angiogenesis. RAD001 (everolimus) is a novel oral mTOR pathway inhibitor. Recent data suggests that RAD001 has direct effects on tumor cell proliferation and may have antiangiogenic activity due to inhibition of tumoral VEGF production and effects on vascular endothelial and smooth muscle cell biology. We first evaluated the in vivo effects of single agent RAD001 in mice bearing the human MM tumor LAGλ-1. Tumor-bearing mice receiving RAD001 at 3, 10, or 30 mg/kg once daily five times per week (M-F) via oral gavage showed marked inhibition of tumor growth at all doses (P

Description: Vascular endothelial growth factor (VEGF) is an important signaling protein that plays a critical role in vasculogenesis and angiogenesis, and serves as one of the contributors to physiological or pathological conditions that can stimulate the formation of new blood vessels. The uncontrolled growth of new blood vessels is an important contributor to a number of pathological conditions, including multiple myeloma (MM). In support of this, bone marrow angiogenesis has been shown to correlate with disease status and poor prognosis in MM. VEGF also directly induces myeloma cell proliferation. We previously evaluated the effects of single agent mouse/human anti-VEGF antibody G6.31 (Campbell et al, Blood (ASH Annual Meeting Abstracts), Nov 2006) in several of our mouse models of human MM. In this study, we evaluated the effects of the same anti-VEGF antibody in combination with bortezomib or lenalidomide. Severe combined immunodeficient (SCID) mice were implanted into the left superficial gluteal muscle with either a 2.0 – 4.0 mm3 fragment from a patient when she was bortezomib-sensitive, LAGκ-1A, or resistant, LAGκ-1B. The tumors were allowed to grow for 21 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups (n=10 mice/group). Treatment groups consisted of a control IgG antibody or anti-VEGF antibody administered via i.p. injection twice weekly at a dose of 2 mg/kg, bortezomib administered via intravenous injection at a dose of 0.25 or 0.5 mg/kg twice weekly, lenalidomide administered via i.p. injection at a dose of 50 mg/kg daily × 5 per week, anti-VEGF antibody (2 mg/kg) + bortezomib (0.25 or 0.5 mg/kg), and anti-VEGF (2 mg/kg) + lenalidomide (50 mg/kg). Mice receiving the combination therapy of anti-VEGF + bortezomib (0.5 mg//kg) antibody showed marked inhibition of tumor growth and reduction of paraprotein levels compared to mice receiving control antibody. Notably, this combination also produced much more marked anti-MM effects compared to bortezomib treatment alone, anti-VEGF antibody alone, or vehicle alone. This combination was well tolerated. In contrast, mice receiving the combination of anti-VEGF antibody + lenalidomide showed no significant differences in tumor volume or hIgG levels compared to single agent treatment or vehicle alone. The markedly improved anti-MM effects of the combination of bortezomib and anti-VEGF antibody compared to single agent treatment in this in vivo study of human MM is promising, and these results have provided the preclinical rationale for an ongoing randomized multi-center Phase II trial.

Description: Mammalian target of rapamycin (mTOR) is a central cell regulator involved in cell survival, growth and proliferation, and is being targeted for cancer therapy. There are two mTOR complexes, the rapamycin-sensitive mTORc1, and the rapamycin-insensitive mTORc2, both of which are downstream of the PI3K/Akt pathway. Protein Kinase C (PKC) refers to a family of serine/threonine kinases that are involved in cell growth, differentiation, apoptosis and migration, and regulated by mTORc2. We evaluated the effects of rapamycin in combination with several PKC inhibitors on the mTOR and PI3K/Akt pathways, two major routes involved in survival of multiple myeloma (MM) cells. First, we examined the expression of several key proteins involved in the regulation of these pathways. PTEN, a phosphatase that blocks AKT activation by inhibiting its upstream regulator PI3K, was highly expressed in U266 and RPMI8226, but found at much lower levels in MM1S. TSC1 and TSC2, proteins regulated by Akt, were also found at much lower levels in MM1S when compared to both U266 and RPMI8226. In contrast to the low levels of PTEN, TSC1 and TSC2, MM1S contained very high levels of PKCζ, a kinase that was undetectable in both U266 and RPMI8226. Given that TSC1 and TSC2 have been shown to negatively regulate mTOR, and PKCζ has been shown to be downstream of mTORc2, we examined the survival and proliferation of MM cells exhibiting normal and over-expression of PTEN following treatment with the mTORc1 inhibitor rapamycin. To modify PTEN expression, MM1S cells were transfected with the over-expression pCEP4-PTEN vector or the empty pCEP4 plasmid. The cells were treated with rapamycin (100nM) for 4 hours and then exposed for 10–30 minutes to FBS. Next, total protein was analyzed by immunoblot for expression patterns and phosphorylation events. The phosphorylation by mTORc1 of S6K was markedly suppressed in cells treated with rapamycin, independent of PTEN expression levels. Additionally, PKCζ phosphorylation was upregulated after treatment with rapamycin, also independent of PTEN expression levels. Based on these results, we hypothesized that blocking mTORc1 leads to a feedback response that increases the activity of mTORc2, resulting in heightened PKCζ phosphorylation levels which may enhance tumor cell growth. Thus, we investigated the effects of blocking mTORc2 through inhibiting its downstream effector PKC as well as mTORc1 with rapamycin on MM cell growth and survival. To determine this, we used several PKC inhibitors in combination with rapamycin. In vitro, MM1S, U266 and RPMI8226 were equally sensitive to single agent rapamycin (IC50 20μM) and the PKC inhibitors rottlerin (IC50 3μM) and Gö 6976 (IC50 1μM). Combinations of rapamycin with either rottlerin or Gö 6976 both significantly increased the ability to inhibit cell proliferation in all three cell lines. As calculated by the Chou-Talalay method, marked synergistic anti-MM effects were observed with both PKC inhibitors. Based on our in vitro results, we are currently evaluating the combination of rapamycin and rottlerin in vivo using our SCID-hu MM models. These promising results provide the potential for further exploration of this new combination approach for the treatment of MM.

Description: Myeloma survival and proliferation in the bone marrow (BM) depends on the expression of a variety of autocrine and paracrine growth factors, including IL-6, insulin-like growth factor I (IGF-I), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). We recently discovered an additional new autocrine myeloma growth factor, pleiotrophin (PTN). PTN is an 18kD heparin-binding protein normally expressed during early development and downregulated in adults, but aberrant PTN re-expression has been associated with a variety of aggressive solid tumors including neuroblastoma, glioma, melanoma and lung, breast and prostate cancers. We found that interference with PTN using a polyclonal anti-PTN antibody inhibited the proliferation of myeloma cells in vitro and in vivo by inducing cell cycle arrest but not apoptosis. To determine the mechanism by which PTN stimulates myeloma proliferation we analyzed the expression of the known PTN receptors, syndecans 1 (CD138) and 3, the anaplastic lymphoma kinase (ALK) and the receptor tyrosine phosphatase beta/zeta (RPTPβ/ζ) on myeloma cells. The expression of ALK and RPTPβ/ζ has not previously been investigated in any hematologic malignancy. In addition to syndecan 1 we found that a subset of myeloma cell lines and BM mononuclear cells (BMMCs) from myeloma patients are RPTPβ/ζ+ by RT-PCR, Western blot and flow cytometry. Myeloma cells, however, do not express ALK. RPTPβ/ζ inhibition by PTN binding leads to the accumulation of β-catenin, and downstream activation of Wnt, NF-κB, MAPK and Akt-mediated cell signals in cells from solid tumors, signaling pathways known to contribute to myeloma cell survival and proliferation. The myeloma cell line MM-1S and the SCID-hu myeloma model LAGλ-1 are both CD138+ but RPTPβ/ζ−. Nonetheless, the growth of these cells is inhibited by anti-PTN antibody. CD138 lacks cytoplasmic signaling motifs suggesting that additional novel receptors are required for PTN-stimulated cell growth in these cells. To continue to define PTN-mediated signaling in myeloma cells we compared the induction of PTN-regulated signaling pathways in RPTPβ/ζ+ RPMI 8226 cells compared to RPTPβ/ζ − MM-1s cells by phospho-protein Western blot. We found differences in PTN-stimulated tyrosine phosphorylation between RPTPβ/ζ+ and RPTPβ/ζ −cells. Specifically, we also discovered that RPTPβ/ζ+ RPMI 8226 cells activate the MAPK Erk1/2. In contrast, we found Akt, but not Erk1/2, to be activated by PTN in RPTPβ/ζ − MM-1S cells. We are now continuing our analysis of PTN signaling in these cells. We are using also identifying new PTN receptors on the surface of myeloma cells by affinity chromatography using biotinylated-PTN. These studies will provide potential new targets that should lead to the development of novel targeted anti-myeloma therapies.