Rational Approaches to Design of Therapeutics
`Targeting Molecular Markers
`
`Richard J. Klasa, Alan F. List, and Bruce D. Cheson
`
`This paper introduces novel therapeutic strategies
`focusing on a molecular marker relevant to a
`particular hematologic malignancy. Four different
`approaches targeting specific molecules in unique
`pathways will be presented. The common theme
`will be rational target selection in a strategy that
`has reached the early phase of human clinical trial
`in one malignancy, but with a much broader poten-
`tial applicability to the technology.
`In Section I Dr. Richard Klasa presents preclini-
`cal data on the use of antisense oligonucleotides
`directed at the bcl-2 gene message to specifically
`downregulate Bcl-2 protein expression in non-
`
`Hodgkin’s lymphomas and render the cells more
`susceptible to the induction of apoptosis.
`In Section II Dr. Alan List reviews the targeting
`of vascular endothelial growth factor (VEGF) and its
`receptor in anti-angiogenesis strategies for acute
`myeloid leukemia (AML) and myelodysplastic
`syndromes (MDS).
`In Section III Dr. Bruce Cheson describes
`recent progress in inhibiting cell cycle progression
`by selectively disrupting cyclin D1 with structurally
`unique compounds such as flavopiridol in mantle
`cell lymphoma as well as describing a new class of
`agents that affect proteasome degradation pathways.
`
`I. ANTISENSE OLIGONUCLEOTIDES DIRECTED AT THE
`BCL-2 GENE MESSAGE IN NON-HODGKIN’S LYMPHOMA
`
`Richard J. Klasa, MD*
`
`Hematologic malignancies in general and non-Hodgkin’s
`lymphomas (NHLs) in particular are frequently associ-
`ated with gain of function mutations, many character-
`ized by balanced chromosomal translocations. Genome-
`wide surveys of gene expression are identifying both
`known and new transcripts that are overexpressed in dif-
`ferent histological subtypes of lymphoma.1 As we de-
`velop molecular classifications of these diseases it is
`assumed that a few key genes will account for the par-
`ticular survival advantage conferred on malignant lym-
`phocytes as compared to their normal counterparts. These
`genes and their protein products would provide rational
`targets for the development of therapeutic strategies to
`reverse this upregulation associated with the malignant
`phenotype.
`
`* Division of Medical Oncology, British Columbia Cancer
`Agency, 600 West 10th Avenue, Vancouver BC V52 4E6
`Canada
`
`Dr. Klasa is on the international advisory boards of Schering
`AG and Hoffmann-LaRoche, and is on the speakers’ bureau
`and advisory board for Berlex Canada.
`
`Non-Hodgkin’s Lymphoma and Bcl-2
`Over the past quarter century cytogenetic analysis has
`identified a number of reciprocal translocations that fre-
`quently occur in histologically identifiable subtypes of
`NHL.2 The transposition of the bcl-2 gene to the immu-
`noglobulin heavy chain promoter region in the t(14;18)
`translocation is associated with > 90% of follicular lym-
`phomas (FL) at diagnosis and 10% of diffuse large B-
`cell lymphomas (DLBCL), making it the most frequent
`event identified in NHL. Additionally, 50% of DLBCL
`overexpress the BCL-2 protein through other mecha-
`nisms, such as gene duplication, and are associated with
`a poorer prognosis after anthracycline-based combina-
`tion chemotherapy.3 BCL-2 is also overexpressed in
`mantle cell lymphoma (MCL), chronic lymphocytic leu-
`kemia (CLL), multiple myeloma (MM) and acute myel-
`ogenous leukemia (AML).4 This same widespread pat-
`tern of distribution is also seen in a variety of solid tu-
`mors including melanoma, small cell lung carcinoma,
`and colon carcinoma as well as prostate and breast car-
`cinoma, especially once the last two are hormone inde-
`pendent. The obvious conclusion is that BCL-2
`overexpression, by whatever means, confers a funda-
`mental advantage to malignant cells and that disruption
`of this overexpression might have therapeutic potential.
`Bcl-2 is an anti-apoptotic member of a large family
`of genes involved in the regulation of programmed cell
`death.5,6 Pro-apoptotic (BAX and BCL-Xs) and anti-
`
`Hematology 2001
`
`443
`
`DR. REDDY’S LABS., INC. EX. 1004 PAGE 1
`
`

`

`apoptotic (BCL-2, BCL-XL) molecules reside within the
`inner mitochondrial membrane and can homo- and
`heterodimerize upon appropriate stimulus. These inter-
`actions control the release of substances such as cyto-
`chrome C from the mitochondria into the cytosol through
`the opening or closing of specific pores in the membrane,
`with permeability determined by the relative abundance
`of the different molecules. Cytochrome C is central to
`the activation of caspases that initiate the apoptotic pro-
`cess. Thus, an overabundance of BCL-2 can prevent or
`retard activation of the apoptotic machinery and allow
`survival under conditions that might otherwise be lethal
`to a cell (Table 1).
`
`Antisense Oligonucleotide
`Reverse complementary or “antisense” oligonucleotides
`(ASOs) are short sequences of single stranded deoxyri-
`bonucleotides complementary to the coding regions of
`a gene that are designed to hybridize by Watson-Crick
`base pairing to messenger-RNA (m-RNA) sequences and
`thus facilitate their degradation.7,8 Naturally occurring
`antisense sequences have been identified as regulators
`of gene expression in a number of systems, supporting
`their potential for therapeutic development.9,10 The for-
`mation of a heteroduplex of m-RNA with the DNA of
`the ASO engages RNaseH, an enzyme that proceeds to
`specifically cleave off the m-RNA moiety, destroying
`the message and putatively leaving the therapeutic ASO
`molecule able to hybridize to another message se-
`quence.11 This results in a reduction in the target m-RNA
`pool, which subsequently leads to reduction in the spe-
`cific protein encoded (Figure 1; see color page 551).
`The presence of the ASO may also prevent the m-RNA
`from appropriately docking with the ribosomal machin-
`ery that would allow translation into a functional pro-
`tein. The end result is loss of expression of that protein
`in the cell.
`ASOs of 16-24 bases in length provide target speci-
`ficity while shorter or longer sequences can result in ran-
`dom hybridization within the transcript repertoire. Se-
`lecting the target areas within a messenger RNA must
`ultimately take into account its tertiary structure, which
`will determine the accessibility of an area for hybridiza-
`
`Table 1. Properties of BCL-2.
`
`• Oncogenic protein
`• Anti-apoptotic
`• Mitochondrial, endoplasmic reticulum, nuclear membrane
`localizations
`• Homotypic and heterotypic dimerization within family
`• Membrane channel/pore function
`• Cytochrome C release from mitochondria via BCL-2 family
`channels regulates cell fate under stress
`
`tion. These target areas are defined in oligonucleotide
`arrays where the entire antisense sequence to an m-RNA
`is displayed in overlapping segments on a slide. The in-
`tensity of hybridization of the labeled message deter-
`mines the candidate therapeutic ASOs.12 Screening of
`oligonucleotide libraries has also identified RNA sites
`that are most accessible to hybridization and correlated
`these sites with protein downregulation and biological
`function.13,14 More empirically, the first 6 codons of the
`open reading frame downstream of the AUG start site
`have repeatedly been found to be accessible to hybrid-
`ization and have been chosen for initial development of
`ASOs against a number of genes.
`As organisms have developed a sophisticated sys-
`tem for dealing with rogue strands of DNA both inside
`and outside the cell, the development of therapeutic
`molecules required chemical modifications to confer
`nuclease resistance and a favorable pharmacokinetic
`profile.15,16 Substitutions in the phosphodiester linkage
`of the bases in the ASO backbone has yielded a number
`of molecules now in clinical development with phospho-
`rothioates being the most widely studied first genera-
`tion molecules (Figure 2). The sulfur substitution yields
`an ASO that is nuclease resistant and capable of enter-
`ing the cell. It demonstrates good hybridization kinetics
`and has little in the way of non-sequence-dependent ef-
`fects or toxicities at concentrations required to down-
`regulate the target message. Additionally, although in
`tissue culture a delivery system such as cationic lipid is
`required for efficient intracellular penetration of these
`highly charged molecules, in vivo ASOs have been
`shown to be active in free form, possibly due to interac-
`tion with blood lipoproteins.17,18
`The correlation of a biologic effect with the spe-
`cific downregulation of target message and protein in
`vivo has been a major focus of the development of ASOs.
`However, ASOs can be very potent immune stimulators,
`by virtue of unmethylated CpG motifs presented in the
`context of certain flanking sequences, and therapeutic
`
`Figure 2. Phosphodiester substitutions in first generation
`antisense oligonucleotides.
`
`444
`
`American Society of Hematology
`
`DR. REDDY’S LABS., INC. EX. 1004 PAGE 2
`
`

`

`Antisense (G3139)
`Reverse Polarity (G3622)
`Mismatch (G4126)
`
`(AS)
`(RP)
`(MM)
`
`5′ tct ccc agc gtg agc cat
`5′ tac cga gtg cga ccc tct
`5′ tct ccc agc atg tgc cat
`
`this as background, we set out to test the in vivo combi-
`nation of ASOs targeting bcl-2 with a cytotoxic agent
`commonly used in the treatment of lymphoma.
`
`Figure 3. Oligodeoxynucleotides targeting Bcl-2.
`
`activity could in part be attributed to nonspecific sys-
`temic immune effects rather then to a specific ASO/
`mRNA interaction.19-22 Additionally, structures such as
`guanosine quartets can demonstrate sequence specific
`but non-antisense biological activity in vitro.23,24 Experi-
`mental designs therefore strive, by the use of appropri-
`ate control oligonucleotides (sense, missense, one and
`two base mismatch mis-sense) and various strains of
`immunodeficient animals, to isolate effects that can be
`attributed to the specific downregulation of target mes-
`sage and protein (Figure 3).
`
`ASOs to bcl-2 and Chemotherapy
`Escalating doses of G3139, cyclophosphamide and the
`combination of both agents were evaluated in severe
`combined immunodeficiency (SCID) mice bearing a sys-
`temic human DoHH2 lymphoma xenograft.35 Experi-
`ments confirmed that G3139 was able to downregulate
`BCL-2 expression in vitro and that treatment with G3139
`alone resulted in prolongation of median survival and
`cure of some animals. (Figure 5, left panel). This effect
`was dose and schedule dependent with no long-term sur-
`vivors seen when a dose of 5mg/kg was given daily for
`14 consecutive days as opposed to > 40% when the dose
`was increased to 12.5 mg/kg on the same daily schedule
`or either 5 or 12.5 mg/kg were administered for 14 treat-
`
`Figure 4. Targeting Bcl-2 may promote apoptosis following
`chemotherapy and irradiation.
`
`ASOs directed at bcl-2
`An 18-mer phosphorothiolated oligonucleotide, G3139,
`directed against the first six codons of the open reading
`frame of the bcl-2 gene message has been developed by
`Genta Pharmaceuticals (Figure 3). Studies of G3139 uti-
`lizing the BCL-2 overexpressing lymphoma cell lines
`DoHH-2 and SU-DHL-4 in vitro have shown down-
`regulation of message and protein expression.25 Tumor
`xenograft models in SCID mice have demonstrated thera-
`peutic activity that is specific when compared to control
`animals as well as animals treated with reverse polarity
`sense, 2-base mismatch mis-sense and non-sense oligo-
`nucleotides.25,26 Extensive pharmakokinetic as well as tox-
`icity studies have been performed identifying a dose range
`with a good therapeutic index.15,16 These findings supported
`the development of clinical trials using G3139 alone as
`treatment for BCL-2 overexpressing follicular lymphomas.
`Further therapeutic potential is sug-
`gested by in vitro experiments confirming
`that bcl-2 plays a major role in the response
`of malignant cells to various stresses which
`produce cellular damage, including chemo-
`therapy (Figure 4).4,27,28 Malignant cell lines
`transfected with a bcl-2 gene with result-
`ant overexpression of the protein product
`demonstrated increased resistance to vari-
`ous chemotherapeutic agents.29-32 Addition-
`ally, cell lines overexpressing BCL-2 were
`rendered more sensitive to killing by che-
`motherapeutic agents when either antisense
`oligonucleotides directed at the bcl-2 mes-
`sage were introduced into the culture or the
`cells were transfected with a vector bear-
`ing the antisense sequence.33,34 This has
`been correlated with a demonstration of
`downregulation of bcl-2 expression. With
`
`Figure 5. Survival of cohorts of 6 mice treated with oligonucleotides alone (left
`panel) or with cyclophosphamide (CY) (right panel).
`All six surviving animals treated with cyclophosphamide and G3139 sacrificed at 90
`days with no molecular evidence of disease detected.
`Saline=control animals ; AS=antisense oligonucleotide G3139 directed at bcl-2 ;
`SN=reverse sequence sense control ; MM= 2-base mismatch control.
`
`Hematology 2001
`
`445
`
`DR. REDDY’S LABS., INC. EX. 1004 PAGE 3
`
`

`

`ments on alternate days (28-day schedule). Similarly,
`cyclophosphamide treatment alone resulted in no long-
`term survivors at lower doses but was able to cure ani-
`mals at high doses. The addition of G3139 to low dose
`cyclophosphamide resulted in the cure of the majority
`of animals (Figure 5, right panel). The interaction be-
`tween the two agents does show dose-response correla-
`tions; for the two low doses of cyclophosphamide tested,
`increasing the dose of G3139 from 2.5 to 5 mg/kg re-
`sulted in longer median survivals and overall increase in
`long-term survivors. A rather striking result was achieved
`when a completely ineffective dose of cyclophospha-
`mide (15 mg/kg, median survival 36 days and no long-
`term survivors) was combined with a modestly effective
`dose of G3139 (2.5 mg/kg, 61 day median survival and
`16% long-term survivors) to produce a 72-day median
`survival and 50% long-term survivors. Mice sacrificed
`at 90 days showed no histological evidence of any dis-
`ease in all tissues analyzed, including immunoperoxidase
`staining for BCL-2, or molecular detection of bcl-2, by
`PCR. This suggests that chemotherapy at very modest
`doses could be made much more effective without in-
`creasing toxicity when combined with an antisense oli-
`gonucleotide. Such an increase in the efficacy of cur-
`rently available agents could significantly alter the prog-
`nosis of a large number of moderately chemotherapy
`sensitive human tumors, resulting in longer median sur-
`vivals and increasing the potential for cure.
`The model has direct relevance to the clinical situa-
`tion faced in NHL, where patients typically present with
`a chemotherapy-sensitive tumor at diagnosis that re-
`gresses only to recur within months to years post-treat-
`ment. The DoHH2 cell line was derived from a follicu-
`lar lymphoma carrying a t(14;18), which results in bcl-2
`gene overexpression. The aggressive nature of the dis-
`ease in this model is, however, more suggestive of a trans-
`formation to a higher-grade histology, a common event
`in follicular lymphoma. Indeed, a recent re-exploration
`of the molecular and cytogenetic features of the cell line,
`using more sensitive detection techniques, has revealed
`a second translocation involving the c-myc oncogene
`with a resultant derivative chromosome 8 carrying
`t(8;14;18).36 We have recently described this phenom-
`enon of double translocation of both bcl-2 and c-myc in
`a subset of patients with small non-cleaved cell (Burkitt-
`like) lymphoma, which represents a very aggressive form
`of the disease.37
`
`Clinical Studies
`G3139 has been studied as a single agent in a phase 1
`trial in heavily pretreated (median of 4 prior regimens)
`patients with relapsed NHL.38,39 Twenty-one patients with
`follicular (9), small lymphocytic (8), diffuse large B-
`cell (3) or mantle cell (1) lymphomas that expressed
`
`BCL-2 were treated at 8 dose levels ranging from 4.6 to
`195.8 mg/m2/day by continuous subcutaneous infusion
`for 14 consecutive days. No significant toxicity was seen
`up to doses of 110 mg/m2/day. One complete and 2 mi-
`nor responses as well as 9 disease stabilizations were
`seen in this heavily pretreated group. BCL-2 protein was
`decreased in 7 out of 16 samples examined, including 2
`from accessible tumor sites and 5 samples of peripheral
`blood or marrow mononuclear cells.
`One study combining a chemotherapeutic agent with
`G3139 has been reported in metastatic melanoma,40 and
`studies are ongoing in a number of other solid tumors
`(melanoma, prostate carcinoma) and hematological ma-
`lignancies (myeloma, chronic lymphocytic leukemia, and
`acute myeloid leukemia). A phase 1 study at our institu-
`tion in relapsed follicular lymphomas combining esca-
`lating doses of both cyclophosphamide and G3139 has
`not identified any unexpected toxicity. The last patient
`enrolled has received cyclophosphamide 750 mg/m2 with
`2.3 mg/kg/day of G3139 by continuous intravenous in-
`fusion for 14 consecutive days (Table 2).
`
`Conclusions
`The identification of overexpression or aberrant expres-
`sion of genes that result in a gain of function, through
`genome wide surveys of cells and tissues in varying
`states, will provide unprecedented insight into the biol-
`ogy of hematological malignancies. Specific down-
`regulation of such overexpression with antisense oligo-
`nucleotides allows disruption of single gene function at
`the messenger RNA and protein level and the study of
`downstream events in the involved molecular pathways
`both in vitro and in vivo. Genes that are critical to the
`differential growth and survival advantage enjoyed by
`malignant cells are being identified and are logical thera-
`peutic targets. The development of ASOs directed at the
`bcl-2 gene provides a model by which a systemic therapy
`for a metastatic disease has been taken from the labora-
`tory through preclinical studies to early phase clinical
`trials, building on knowledge of this particular gene’s
`role in cellular apoptosis. Combining multiple antisense
`
`Table 2. Phase I study of G3139 and cyclophosphamide in
`relapsed follicular lymphoma.
`
`G3139 mg/kg/day Cyclophosphamide
`mg/m2/IV
`CIVI
`(day 1 to 14)
`(day 8)
`0.6
`250 done
`1.2
`250 done
`2.3
`250 done
`2.3
`500 done
`2.3
`750 ongoing
`3.1
`1000
`5.0
`1000
`
`Level
`1
`2
`3
`4
`5
`6
`7
`
`446
`
`American Society of Hematology
`
`DR. REDDY’S LABS., INC. EX. 1004 PAGE 4
`
`

`

`strategies with other therapeutic modalities has the po-
`tential to increase the specificity of the treatments avail-
`able to our patients, thus improving their efficacy and
`reducing toxicity.
`
`II. TARGETING ANGIOGENESIS IN HEMATOLOGIC
`MALIGNANCIES
`
`Alan F. List, MD*
`
`The seminal observations that the growth and metastatic
`potential of solid tumors is dependent upon the forma-
`tion of new blood vessels triggered an enormous expan-
`sion in angiogenic research that has yielded novel thera-
`peutics targeting an array of angiogenic molecules. In-
`vestigations of the relevance of angiogenesis in hemato-
`logic malignancies are still at an early stage, but accu-
`mulating evidence indicates that the angiogenic profile
`of many hematologic malignancies is distinct from that
`of solid tumors. As progeny of a common endothelial
`and hematopoietic stem cell, hematologic malignancies
`may elaborate and respond to angiogenic factors in a
`paracrine or autocrine fashion, contributing to tumor cell
`survival and expansion, adhesion, bone resorption and
`immune suppression.
`
`Angiogenesis
`Blood vessel development is characterized by two dis-
`tinct biologic processes, vasculogenesis, and angiogen-
`esis. Vasculogenesis, which is largely restricted to em-
`bryonic development, involves de novo endothelial cell
`differentiation from mesodermal precursors as the pre-
`requisite for coordinated blood vessel generation.1 An-
`giogenesis, the process of new blood vessel formation
`from preexisting vessels, is responsible for the genera-
`tion of neovasculature in adult life, and occurs physi-
`ologically during wound healing and within female re-
`productive organs during the menstrual cycle, as well as
`in pathologic conditions such as proliferative retinopa-
`thy, arthritic synovium, and human malignancies.2 An-
`giogenesis is a multistep process that includes both acti-
`vation and resolution phases. The activation phase is
`responsible for the sequential events of basement mem-
`brane degradation, endothelial cell proliferation and
`migration, and capillary lumen formation. The resolu-
`tion phase is responsible for the maturation and stabili-
`zation of the newly formed microvasculature through
`the recruitment of pericytes, promotion of basement
`membrane reconstitution, and subsequent extinction of
`the endothelial cell mitogenic response. A large number
`of pro-angiogenic molecules and endogenous angiogen-
`
`* Arizona Cancer Center, 1515 N Campbell, Room 3945, PO
`Box 245004, Tucson AZ 85774
`
`esis inhibitors that coordinate the angiogenic response
`have been delineated (Table 3).3 Vascular endothelial
`growth factor (VEGF), first identified in 1989 and later
`isolated from the HL-60 myeloid leukemia cell line, is a
`critical regulator of vascular development that is respon-
`sible for activation of endothelial cell proliferation dur-
`ing vasculogenesis and the direction of capillary sprout-
`ing during angiogenesis.4 Indeed, gene inactivation stud-
`ies indicate that VEGF is essential to the neoplastic an-
`giogenic response.5
`
`VEGF and Receptor Tyrosine Kinases
`The VEGF-A molecule is a disulfide-linked homodimer
`represented by five different isoforms generated by al-
`ternate exon splicing of gene message.6 The correspond-
`ing VEGF monomers range in size from 121 to 206
`amino acids, with smaller molecules (i.e., 121, 145, and
`165 amino acids) representing the secreted and diffusable
`isoforms, whereas the larger proteins (189, 206 amino
`acids) are sequestered by heparin sulfate residues present
`on cell surfaces or within the extracellular matrix.7,8 Al-
`though all isoforms are biologically active, the VEGF165
`isoform predominates and is recognized as a more po-
`tent and bioavailable endothelial cell mitogen.9,10 Recent
`investigations indicate that the VEGF family is composed
`of five members in addition to the prototype, VEGF-A,
`including VEGF-B, VEGF-C, VEGF-D, VEGF-E and
`PIGF (placental growth factor).6 Within the arterial wall,
`VEGF is produced by smooth muscle cells in response
`to oxidative stress and other stimuli.11 Autocrine pro-
`duction of VEGF and corresponding receptor upregu-
`lation is also demonstrable in endothelial cells in response
`to hypoxia, nitric oxide, VEGF deprivation and other
`cellular stresses.12,13
`Trophic response to the VEGF family members is
`directed by selective interaction with structurally ho-
`mologous type III receptor tyrosine kinases (RTKs), in-
`cluding VEGFR-1, originally termed fms-like tyrosine
`kinase (FLT-1),14 VEGFR-2 or kinase insert domain-con-
`taining receptor (KDR)/fetal liver kinase-1 (flk-1),15 and
`the recently characterized VEGFR-3 or FLT-4 receptor
`(Figure 6).16 Each of these receptors contains seven ex-
`tracellular immunoglobulin homology domains that cre-
`ate the ligand-binding site, a single short transmembrane-
`spanning sequence, and a cytoplasmic tail that contains
`the tyrosine kinase domain akin to that of the c-kit and
`PDGF-receptors.17,18 The external ligand-binding com-
`ponent of VEGFR-3 differs from the other VEGF re-
`ceptors in that the fifth immunoglobulin domain is
`cleaved during receptor processing to yield covalent,
`disulfide-linked subunits.19 In adults, VEGFR-1 and
`VEGFR-2 expression is limited to the vascular endothe-
`lium, monocytes (VEGFR-1), and primitive hematopoi-
`etic precursors (VEGFR-2), whereas VEGFR-3 is re-
`
`Hematology 2001
`
`447
`
`DR. REDDY’S LABS., INC. EX. 1004 PAGE 5
`
`

`

`Table 3. Endogenous stimulators and inhibitors of angiogenesis.
`
`Native Inhibitors
`Interferon (IFN)-α,γ
`
`Prolactin (16 Kd fragment),
`IFN-α,γ
`
`Tissue inhibitor of
`metalloprotease
`(TIMP-1, TIMP-2)
`Plasminogen activator-
`inhibitor-1 (PAI-1)
`Zinc
`p53 Rb
`
`Angiogenic Molecules
`VEGF (A-D), PIGF
`Angiogenin
`Angiotropin
`Epidermal growth factor (EGF)
`Fibroblast growth factor
`(acidic and basic; FGF)
`Hepatocyte growth factor/scatter
`factor (HGF, SF)
`Platelet-derived growth factor (PDGF)
`Tumor necrosis factor-α (TNFα)
`Interleukin-8 (IL-8)
`Insulin-like growth factor-1 (IGF-1)
`Cathepsin
`Proteases and
`Protease Inhibitors Gelatinase-A, -B (MMP2,9)
`Stromelysin
`Urokinase-type plasminogen
`activator (uPA)
`Copper
`c-myc
`ras
`c-src
`v-raf
`c-jun
`Signal Transduction Thymidine phosphorylase
`Enzymes
`RAS - arnesyl transferase
`Geranylgeranyl transferase
`Interleukin-1
`Interleukin-6
`Interleukin-8
`α
`β
`3 integrin
`v
`Angionpoietin-1 (Ang-1)
`Antionstatin II (AT1 receptor)
`Endothelin (ETB receptor)
`Erythropoietin
`Hypoxia
`iNOS
`Platelet-activating factor (PAF)
`Prostaglandin E, COX-2
`Thrombopoietin
`
`regulate extracellular matrix (ECM)
`degradation and endothelial cell migra-
`tion via protease elaboration.31 VEGF-
`E is an alternate ligand for VEGFR-2
`and has biologic activities similar to
`VEGF-A.32 Neuropilin-1 (NRP-1), a
`cell surface glycoprotein, acts as a co-
`receptor for VEGF-A165 to enhance its
`binding to VEGFR-2 on endothelial
`cells. Unlike the VEGF receptors, NRP-
`1 lacks an intracellular domain and
`therefore is not a direct mediator of
`cytokine signaling.33
`VEGF-A exerts its biologic effects
`via interaction with either VEGFR-1
`or VEGFR-2. VEGF receptors dimer-
`ize in response to ligand engagement,
`activating the tyrosine kinase located on
`the intracellular cytoplasmic tail. The
`resultant auto-phosphorylation of these
`receptors on specific tyrosine residues
`creates docking sites for cytoplasmic
`signaling molecules.34 Activation of
`VEGF receptors in endothelial cells trig-
`gers recruitment of adaptor and signal-
`ing proteins that contain Src homology
`domain-2 (Sh-2) including Shc and
`Sck.35,36 Recruitment and phosphoryla-
`tion of these adaptor molecules permit
`their binding to the p85 subunit of phos-
`phatidyl inositol-3 (PI-3) kinase and to
`phospholipase-Cg, activating the PI-3
`kinase and ERK (stress activated pro-
`tein kinase-1, 2) kinase signaling cas-
`cades.37-39 PI-3 kinase phosphorylates
`Akt (protein kinase-B), a serine/threo-
`nine kinase involved in antiapoptotic
`signaling.40 Akt activation permits its
`translocation to the plasma membrane,
`where it phosphorylates and inactivates a variety of pro-
`apoptotic molecules, including BAD, caspase-9, mito-
`chondrial Raf, and the forkhead transcription factor-1
`(FKHR-1).41 Similarly, through PI-3 kinase activation,
`VEGF-A activates focal adhesion kinase in endothelial
`cells, a step that is critical to the recruitment and activa-
`tion of cell adhesion molecules containing β
`
`1, β2, and β
`3
`integrins.42,43 Indeed VEGFR-2 interaction with its ligand
`triggers receptor association with PI-3 kinase, vascular
`endothelial (VE)-cadherin and α
`β
`3, components that
`V
`are essential to the creation of an active multimeric sig-
`naling complex. Thus, VEGFR-2 activation assures
`proper juxtaposition of the receptor with cytoskeletal
`proteins.44,45
`
`Growth factors
`
`Trace Elements
`Oncogenes
`
`Cytokines
`
`Endogenous
`
`Interleukin-10
`Interleukin-12
`
`Angiopoietin-2
`Angiotensin
`Angiotensin II (AT2 receptor)
`Caveolin-1, caveolin-2
`Endostatin
`Interferon-α
`Isoflavones
`Platelet factor-4
`
`Thrombospondin
`Troponin-1
`
`Abbreviations: VEGF, vascular endothelial growth factor; PIGF, placental growth factor;
`iNOS, inducible nitric oxide synthetase; MMP, matrix metalloproteases
`
`stricted to lymphatic endothelium.20-24 The critical role
`for these receptors and that of VEGF has been demon-
`strated in gene inactivation studies. VEGF-A and the
`VEGFR-2 receptor are essential to embryonic vascu-
`logenesis and definitive hematopoiesis.25-27 In vitro in-
`vestigations indicate that VEGFR-2 signaling is respon-
`sible for proliferation and differentiation of endothelial
`cells in response to VEGF-A stimulation, whereas
`VEGFR-1 has been implicated as a decoy receptor and
`potential activator of endothelial permeability.28 Ligand
`binding to VEGFR-3 is restricted to VEGF-C and VEGF-
`D, which together direct lymphangiogenesis.17,29,30 The
`actions of the remaining VEGF family members are less
`well-defined; however, receptor recognition for VEGF-
`B appears restricted to VEGFR-1 where it is believed to
`
`448
`
`American Society of Hematology
`
`DR. REDDY’S LABS., INC. EX. 1004 PAGE 6
`
`

`

`transcriptional activator of other genes in-
`cluding erythropoietin, inducible nitric ox-
`ide synthetase (iNOS), insulin growth fac-
`tor (IGF-2), glycolytic enzymes, and tumor
`growth factor-β (TGF-β).53
`
`Hemangioblast and Hematopoiesis
`During embryogenesis, pluripotential he-
`matopoietic stem cells originate from cell
`clusters in the ventral floor of the pre-um-
`bilical dorsal aorta.54 These common he-
`matopoietic/endothelial (HE) precursors
`share an antigenic phenotype characterized
`by expression of the progenitor cell antigen,
`CD34, and VEGFR-2.27,55 Murine gene
`knockout studies indicate that VEGFR-2 ex-
`pression is required for vasculogenesis and
`for establishment of definitive hematopoie-
`sis. Although HE progenitors can be gener-
`ated in vitro from VEGFR-2 deficient em-
`bryonal stem cells, VEGFR-2 signaling is
`essential for progenitor migration and endot-
`helial commitment. Post-natally, heman-
`gioblasts, or HE progenitors, persist and con-
`tribute to long-term hematopoiesis and to the
`generation of circulating endothelial cells.
`Confirmation of leukemia specific gene ex-
`pression (i.e., bcr/abl in chronic myeloid leu-
`kemia) and the identification of donor-spe-
`cific DNA alleles (i.e., in allograft recipi-
`ents) within vascular endothelium provides definitive
`proof that bone marrow-derived hemangioblast progeny
`contribute to the maintenance of endothelium in adults.56-
`58 These primitive hemangioblast progenitors represent
`less than 2% of circulating CD34+ cells and display a
`surface phenotype characterized by CD34+, AC133+,
`VEGFR-2+, platelet endothelial cell adhesion molecule
`(PECAM), and the stromal derived factor-1 (SDF-1) re-
`ceptor, CXCR-4.59 Under appropriate culture conditions,
`hematopoietic commitment of these precursors is demon-
`strable by acquisition of the leukocyte common antigen,
`CD45, and β1 integrins, whereas angioblasts display VE-
`cadherin.
`VEGF receptorexpression extends beyond the primi-
`tive progenitor compartment during adult hematopoie-
`sis. VEGFR-1 is expressed by monocytes and commit-
`ted CD34+ progenitors, and is responsible for activation
`of migration and maturation skewing, respectively.22,23,60
`With the exception of primitive hematopoietic progeni-
`tors, VEGFR-2 expression appears restricted to mega-
`karyocytes.20 Within the adult bone marrow, cellular
`expression of angiogenic molecules outside of stromal
`elements is restricted to erythroblastic islands. Both
`VEGF-A and PIGF are secreted by erythroblasts within
`
`Figure 6. Interaction of vascular endothelial growth factor (VEGF) family
`members with their cognate receptors on endothelial cells.
`The VEGF receptors harbor seven extracellular immunoglobulin (Ig)-homology
`domains. The fifth Ig domain of VEGFR-3 is cleaved after biosynthesis to yield a
`stable disulfide bond linking the Ig subunits. VEGFR-1 and VEGFR-2 initiate signals
`essential to the angiogenic response, whereas VEGFR-3 regulates lymph angiogen-
`esis. NRP-1 (neuropilin-1) binds to the carboxy-terminal sequence of VEGF165 to
`enhance binding of the angiogenic molecule to the VEFGR-2 receptor.
`Abbreviations: TK, tyrosine kinase; MMP, matrix metalloproteases; PIGF, placental
`growth factor.
`
`VEGF Regulation
`Transcriptional regulation of VEGF is influenced by a
`number of signals that converge upon the oxygen sensi-
`tive transcription factor, hypoxia-inducing-factor (HIF)-
`1a. HIF is a heterodimeric protein composed of α and β
`subunits.46 The β subunit is expressed constitutively,
`whereas the a subunit is a member of a family of DNA-
`binding proteins that contain trans-activation domains
`within the carboxy-terminus. Under normal oxygen con-
`ditions, HIF-1a is degraded rapidly by the ubiquitin-
`proteasome pathway.47 Hypoxia stabilizes the HIF-1a
`protein by inhibiting its proteasome degradation. The
`von Hippel-Lindau (VHL) tumor suppressor gene is one
`component of the ubiquitin ligase complex that
`ubiquinates and promotes the degradation of HIF-1a
`under normoxic conditions. Inactivation of VHL, as oc-
`curs in the von Hippel-Lindau syndrome, stabilizes HIF-
`1a and sustains tumor production of VEGF.48 Inactiva-
`tion of a negative regulator of HIF-1a such as VHL is
`only one of many signals which converge upon HIF-1a
`to activate VEGF expression. Alternate signals include
`sustained RAS activation, β-integrin activation, stress-
`induced ERK signaling, and inactivation of p53.49-52 In
`addition to its regulatory effects on VEGF, HIF-1a is a
`
`Hematology 2001
`
`449
`
`DR. REDDY’S LABS., INC. EX. 1004 PAGE 7
`
`

`

`erythroid islands,61 suggesting that