ALBERT

Description: Multitargeted treatment approaches have been shown to be more effective than single agent therapy in multiple myeloma (MM). In addition, agents targeting not only the MM cells directly but also their microenvironment, like bone marrow stromal cells (BMSCs), endothelial cells, and osteoclasts (OCLs) causing enhancement of tumor cell growth, angiogenesis, and MM bone disease, respectively, are promising new treatment modalities for this still non-curable disease.Here we investigated the novel, orally available multi-kinase inhibitor BAY 73-4506, currently in phase I clinical trials, for its therapeutic effect in MM. BAY is a potent inhibitor of angiogenic (VEGFR 1-3, PDGFR-b), as well as oncogenic, kinases (cKIT, RET, FGFR, Raf). We first tested the ability of BAY to suppress MM cell proliferation and survival in a wide array of MM cell lines (MM.1S, RPMI 8226, NCI H929, OPM2, KMS11, KMS 18, INA6, U266, KMS12BM, S6B45), including those resistant to conventional chemotherapeutics (MM.1R, Dox40, LR5). Our data show that BAY is active in all cell lines tested in a low micromolar range equivalent to concentrations achieved in patient plasma during the first clinical trial in solid tumors. Importantly, BAY also overcomes the growth advantage conferred in a BMSC-MM, as well as an endothelial cell-MM, coculture system. BAY treatment abrogates MEK, ERK and AKT phosphorylation in a time and dose dependent manner, followed by induction of apoptosis, evidenced by Annexin staining and DNA fragmentation. Since VEGF signaling pathway is a potent inducer of angiogenesis and BAY targets VEGFR 1-3, we examined anti-angiogenic properties of BAY. This compound inhibits endothelial cell growth and endothelial cell tubuli formation in vitro at concentrations less than 1mM; moreover, BAY markedly inhibits the VEGF-induced cell migration on fibronectin. Activation of MAP kinase is a critical event during OCL differentiation, activation, and survival; BAY inhibits osteoclastogenesis, evidenced by blockade of M-CSF/RANKL-triggered differentiation of mononuclear cells to TRAP-positive osteoclasts, an important marker of osteoclastogenesis. Finally, combination treatment of BAY with dexamethasone shows synergistic effects on MM cell growth and survival. These in vitro experiments on the effects of BAY on MM tumor cells directly, in co-culture with endothelial or BMSCs, as well as on osteoclast differentiation, provides the basis for its evaluation in a murine model of human MM to confirm these promising in vitro effects of this novel multi-kinase inhibitor, finally leading to clinical evaluation to improve patient outcome.

Description: We have previously shown that the novel orally available small molecule inhibitor of PKC enzastaurin (Eli Lilly and Company) inhibits MM cell growth, survival and angiogenesis both in vitro and in vivo. To date, however, the downstream effects contributing to growth inhibition and cell death remain to be determined. Here, we performed global gene expression profiling on enzastaurin treated MM cells and identified 200 Genes to be differentially regulated with a 〉 2-fold cut off. Strikingly, two major groups of up-regulated probe sets were associated with either of two pathways - endoplasmatic reticulum (ER)-stress response or WNT-signaling. Importantly, MM cells, producing high levels of paraprotein, are highly susceptible to perturbation of ER function and protein folding. Moreover, PKC isoforms have been reported to directly regulate the canonical WNT pathway via phosphorylation of b-catenin (CAT), leading to its ubiquination and proteasomal degradation. Specifically, we fist evaluated the role of enzastaurin in mediating ER-stress in MM cells. The transcriptional up-regulation of genes involved in ER-stress (GADD153/CHOP, GADD34, ATF3), triggered by enzastaurin at 3h, was confirmed by western blot analysis, accompanied by induction of the molecular ER chaperone BiP/grp78, phosphorylation of eIF2a consistent with PERK activation, and up-regulation of p21. These events were preceded by an early (1h) increase of intracellular calcium levels, a hallmark of ER-stress, assessed by FLUO4 staining. These data suggest an important role of ER-stress response in the early growth inhibition of MM cells caused by enzastaurin. Second, we delineated effects of enzastaurin on WNT pathway in MM and other tumor cell lines. Upon enzastaurin treatment, CAT was dephosphorylated at Ser33, 37, 41 in a dose- and time-dependent manner in all cell lines tested (10 MM, 3 colon cancer, HeLa, as well as human embryonic kidney 293 cells). Consequently, accumulation of CAT occurred in both cytosolic and nuclear fractions of treated MM cells, associated with activated TOPflash LUC-reporter system, confirming nuclear transactivating activity. Specific inhibition of CAT by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a CAT-dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; CAT induction by enzastaurin led to p73 (but not p53) activation and was also abrogated by CAT-specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of CAT in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. Our studies therefore indicate that ER-stress response contributes to the immediate inhibition of proliferation by enzastaurin, followed by CAT accumulation leading to p73 activation, contributing to enzastaurin-mediated cell death. These findings provide a novel link between CAT and p53-family members. Moreover p73, which is only rarely mutated in human cancers, represents a novel therapeutic target in MM.

Description: Tissue factor (TF) is a trans-membrane glycoprotein that is the cellular receptor for coagulation factor VII/VIIa. Activation of factor VII by TF is well established as the primary regulator of blood coagulation. Although TF has been implicated in such diverse processes as angiogenesis and tumor metastasis, these other functions have been difficult to study, as the knockout of murine TF is lethal at day 8.4 of embryonic life. Because of their hardiness, their rapid maturation sequence and their optical clarity, the zebrafish is an increasingly popular model for studying embryonic development. In order to study the role of TF in embryonic development we have cloned zebrafish TF cDNA. The 1.36kb orthologous cDNA predicts an ORF encoding a 293 amino acid (aa) protein with a 26 amino acid (aa) signal peptide. The predicted mature protein contains a 230 aa extra-cellular domain, a 20 aa trans-membrane segment, and a 17 aa cytoplasmic tail. The predicted amino acid sequence of zebrafish TF has 31% and 32% overall identity with human and mouse TF respectively. The zebrafish TF gene, like the human and mouse genes, has 6 exons spanning 14kb of genomic DNA. Whole-mount in situ hybridization with a TF anti-sense Riboprobe revealed that TF is detected as early as 18 hours post-fertilization (hpf) in developing head structures and brain. At 20 hpf, TF is expressed in the vascular system and highly expressed in the hindbrain. By 48 hpf, TF is seen in the brain, heart, liver, lens and gut. To investigate the expression of zebrafish TF in vivo, we generated transgenic zebrafish lines that express enhanced green fluorescent protein (EGFP) under the control of the TF promoter. A ∼ 2.0 kb promoter fragment upstream of the first exon of the TF gene was sufficient to drive expression of EGFP in a manner that recapitulated the expression of native TF. We inhibited TF expression by injecting anti-sense oligonucleotide morpholinos into the Tg(fli1:EGFP) line, in which there is robust expression of EGFP in vascular endothelial cells and their angioblast precursors. At 24 hpf the development of trunk capillary beds was delayed and, at 48 hpf, most of the morphants developed pericardial edema and impaired formation of inter-somitic vessels. These studies demonstrate that TF is expressed in multiple organs during zebrafish embryogenesis and that TF expression is necessary for the proper migration of endothelial cells into developing blood vessels during the process of embryonic vasculogenesis.

Description: Myeloid cell leukaemia-1 (Mcl-1) is an anti- apoptotic member of the Bcl-2 family commonly expressed in multiple myeloma (MM). Drugs (e.g. bortezomib) can induce a 28kD Mcl-1 fragment (Mcl-1Δ128–350) in a caspase- dependent manner, which induces inhibition of MM cell proliferation and apoptosis. Here we sought to delineate molecular sequelae downstream of Mcl-1Δ128–350 which mediate its anti- proliferative and pro-apoptotic effects in MM and other malignant cells. Our results demonstrate that exogenous Mcl-1Δ128–350 induces upregulation and nuclear accumulation of c-Jun, as well as generation of a pro-apoptotic 60kD c-Abl fragment (c-Abl Δ). Bortezomib treatment triggered c-Jun upregulation in Mcl-1wt/wt, but not Mcl-1 null, murine embryonic fibroblasts (MEFs), and neither transfection with exogenous c-Jun nor with exogenous 60kD c-Abl Δ triggered the generation of Mcl-1Δ128–350. Moreover, drug-induced generation of Mcl-1Δ128–350 was not abrogated by specific transient knockdown of c-Jun or c-Abl by small interfering RNA, further supporting the requirement of Mcl-1Δ128–350 for c-Jun upregulation. Our studies also identified mechanisms downstream of upregulated c-Jun which trigger inhibition of MM cell proliferation and apoptosis. Interestingly and similar to c-Jun and c-Abl Δ, Mcl-1Δ128–350 accumulates within the nuclear fraction. Indeed, interaction of Mcl-1Δ128–350 with c-Jun, as well as subsequent enhanced AP-1 reporter activity, demonstrate a direct regulatory role of Mcl-1Δ128–350 in c-Jun- dependent gene transcription. Finally, gene profiles in MM cells transfected with either Mcl-1wt or Mcl-1Δ128–350 identify differentially expressed genes associated with MM cell proliferation, survival and drug resistance. Taken together, these data both delineate the role of Mcl-1 in MM pathogenesis and further support targeting Mcl-1 in novel MM treatment strategies.

Description: Agents targeting not only myeloma cells directly but also bone marrow stromal cells (BMSCs), endothelial cells, and osteoclasts (OCLs) that cause enhancement of tumor cell growth, angiogenesis, and MM bone disease, respectively, are promising new treatment modalities for multiple myeloma. Here we investigated the novel, orally available multi-kinase inhibitor BAY 73-4506 (BAY), currently in phase II clinical trials, for its therapeutic effect in MM. BAY is a potent inhibitor of angiogenic (VEGFR 1–3, PDGFR-β), as well as oncogenic, (cKIT, RET, FGFR, Raf) kinases We first tested the ability of BAY to suppress proliferation and survival in a wide array of MM cell lines, including those resistant to conventional chemotherapeutics. Our data show that BAY, in a low micromolar range that is well below concentrations achieved in patient plasma during the first clinical trial in solid tumors, induces apoptosis by caspase-9 and caspase-3 activation in all cell lines tested. Importantly, BAY also overcomes the growth advantage conferred in a BMSC-MM, as well as an endothelial cell-MM, co-culture system. BAY treatment abrogates growth factor-stimulated MEK, ERK and AKT phosphorylation at sub-micromolar concentrations. Since the VEGF signaling pathway is a potent inducer of angiogenesis and BAY targets VEGFR 1–3, we examined its anti-angiogenic properties. BAY inhibits endothelial cell growth and endothelial cell tubule formation in vitro at concentrations less than 1μM; moreover, it also markedly inhibited VEGF-induced cell migration on fibronectin. Activation of MAP kinase is a critical event during OCL differentiation, activation, and survival; and importantly, BAY also inhibits osteoclastogenesis, evidenced by blockade of M-CSF/RANKL-triggered differentiation of mononuclear cells to TRAP-positive osteoclasts. Finally, BAY significantly delays tumor growth and abrogates blood vessel formation in vivo in a xenograft mouse model of human MM. These in vitro and in vivo results provide the basis for further clinical evaluation of BAY to improve patient outcome in MM.

Description: In multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester–induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCμ. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.

Description: In multiple myeloma (MM), protein kinase C (PKC) overexpression has been reported including the prognostic adverse patient group with t (4;14) translocation. Importantly, PKC signaling pathways have been implicated in MM cell proliferation, apoptosis, and migration. Here, we investigated the novel, orally available PKC inhibitor Enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits membrane, cytosolic, and nuclear phosphorylation of homologous PKC isoform residues, as well as associated kinase activity, induced by the major PKC activator TPA (Tumor-promoting phorbol ester). Consequently, it also abrogates TPA-induced phosphorylation of signaling molecules downstream of PKC including MARCKS and PKCm. In MM, Enzastaurin inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs); co-stimulation with the extracellular matrix protein fibronectin, VEGF or IL-6; as well as MM patient serum. Phosphorylation of downstream signaling molecules was also abrogated, including cytoplasmic and nuclear ERK, JNK, ribosomal protein S6 and GSK3b as well as nuclear cMyc. Enzastaurin inhibits both proliferation and survival of MM cell lines and MM cells isolated from multidrug- resistant patients; as well as overcomes MM cell growth triggered by tumor cell binding to BMSCs and endothelial cells at a low micromolar range equivalent to the concentrations achieved in patient plasma during clinical trials. Importantly, synergistic cytotoxicity is observed when Enzastaurin is combined with bortezomib. Besides proliferation and survival, Enzastaurin inhibits MM cell adhesion, as well as VEGF- and IGF-1-triggered MM cell migration. It also blocks VEGF- triggered signaling pathways in endothelial cells, thereby inhibiting tubule formation. Finally and most importantly, tumor growth, survival, and angiogenesis are abrogated by Enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor Enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve patient outcome in MM.

Description: Erythropoietin (Epo), along with its receptor EpoR, is the principal regulator of red cell development. Upon Epo addition, the EpoR signaling through the Janus kinase 2 (JAK2) activates multiple pathways including Stat5, phosphoinositide-3 kinase (PI-3K)/Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor signaling. Here, we showed that Lnk-deficient mice have elevated numbers of erythroid progenitors, and that splenic erythroid colony-forming unit (CFU-e) progenitors are hypersensitive to Epo. Lnk-/- mice also exhibit superior recovery after erythropoietic stress. In addition, Lnk deficiency resulted in enhanced Epo-induced signaling pathways in splenic erythroid progenitors. Conversely, Lnk overexpression inhibits Epo-induced cell growth in 32D/EpoR cells. In primary culture of fetal liver cells, Lnk overexpression inhibits Epo-dependent erythroblast differentiation and induces apoptosis. Lnk blocks 3 major signaling pathways, Stat5, Akt, and MAPK, induced by Epo in primary erythroblasts. In addition, the Lnk Src homology 2 (SH2) domain is essential for its inhibitory function, whereas the conserved tyrosine near the C-terminus and the pleckstrin homology (PH) domain of Lnk are not critical. Furthermore, wild-type Lnk, but not the Lnk SH2 mutant, becomes tyrosine-phosphorylated following Epo administration and inhibits EpoR phosphorylation and JAK2 activation. Hence, Lnk, through its SH2 domain, negatively modulates EpoR signaling by attenuating JAK2 activation, and regulates Epo-mediated erythropoiesis. (Blood. 2005; 105:4604-4612)

Description: Our own and other previous studies demonstrate marked anti-proliferative activity of the tyrophostin adaphostin (NSC680410) in a variety of hematologic malignancies including chronic myelocytic leukemia (CML), chronic lymphcytic leukemia (CLL), acute myelocytic leukemia (AML), and Multiple Myeloma. Here we show that adaphostin (NSC680410), similar to bortezomib, additionally inhibits tumor angiogenesis within the MM bone marrow (BM) microenvironment. This effect is elicited both indirectly by inhibition of VEGF production and secretion in MM cells, as well as directly by abrogation of endothelial cell growth. Specifically, adaphostin triggers marked downregulation of nuclear c-Myc expression in MM cells. Both adaphostin, as well as specific downregulation of c-Myc using siRNA, lead to a decrease in cobalt chloride- induced Hif-1alpha- expression and Hif-1alpha activity, as evidenced by western blot analysis and expression of Hif-1alpha- driven luciferase, respectively. Indeed secretion of the Hif-1alpha target gene VEGF is markedly inhibited in a dose- and time- dependent manner. Importantly, neither knockdown of c-Abl expression nor exogenous overexpression of caspase- cleavage- induced c-Abl fragment abrogates drug- induced Hif-1alpha downregulation or inhibition of its activity. Taken together, these results indicate the existence of a c-Myc/ Hif-1alpha- dependent, but c-Abl- independent, pathway modulating MM cell production and secretion of VEGF. In contrast, we demonstrate a direct antiangiogenic effect of adaphostin on endothelial cells, similar to H2O2, is mediated via c-Jun upregulation, inhibition of cell proliferation, and the induction of cell apoptosis. Moreover, our data further demonstrate activity of adaphostin within the BM microenvironment. Adaphostin, similar to bortezomib, significantly inhibits VEGF secretion triggered by adhesion of MM cells to BMSCs and endothelial cells. Consequently, conditioned medium derived from adaphostin- treated co-cultures markedly inhibits endothelial cell growth and tubule formation in a dose- dependent manner. Finally, we confirmed these in vitro results using an in vivo xenograft mouse model of human MM. Specifically, western blot analysis, as well as immunohistochemistry, demonstrate marked downregulation of both Hif-1alpha and CD31 in tumors isolated from adaphostin- treated animals versus control animals, confirming the in vivo antiangiogenic effect of adaphostin. Similar effects were obtained using a SCIDhu mouse model as well as a significant decrease of MM- related bone disease, due to anti- VEGF activity of adaphostin. Taken together, these data provide the rationale for the clinical evaluation of adaphostin to target both MM cells and the BM milieu to improve patient outcome in Multiple Myeloma.

Description: Targeting protein kinase C (PKC) isoforms by the small molecule inhibitor enzastaurin has shown promising preclinical activity in a wide range of tumor cells. We further delineated its mechanism of action in multiple myeloma (MM) cells and found a novel role of β-catenin in regulating growth and survival of tumor cells. Specifically, inhibition of PKC leads to rapid accumulation of β-catenin by preventing the phosphorylation required for its proteasomal degradation. Microarray analysis and small-interfering RNA (siRNA)–mediated gene silencing in MM cells revealed that accumulated β-catenin activates early endoplasmic reticulum stress signaling via eIF2α, C/EBP-homologous protein (CHOP), and p21, leading to immediate growth inhibition. Furthermore, accumulated β-catenin contributes to enzastaurin-induced cell death. Sequential knockdown of β-catenin, c-Jun, and p73, as well as overexpression of β-catenin or p73 confirmed that accumulated β-catenin triggers c-Jun–dependent induction of p73, thereby conferring MM cell apoptosis. Our data reveal a novel role of β-catenin in endoplasmic reticulum (ER) stress-mediated growth inhibition and a new proapoptotic mechanism triggered by β-catenin on inhibition of PKC isoforms. Moreover, we identify p73 as a potential novel therapeutic target in MM. Based on these and previous data, enzastaurin is currently under clinical investigation in a variety of hematologic malignancies, including MM.