Michael S. Boosalis

Receptor for the globular heads of C1q (gC1q‐R, p33, hyaluronan‐binding protein) is preferentially expressed by adenocarcinoma cells

Combinatorial Ig libraries with phage display allow in vitro generation of human Ig fragments without the need to maintain hybridomas in ongoing cell culture or to select circulating Ig from human serum. Identifying tumor‐associated antigens on the surface of intact tumor cells, as opposed to purified proteins, presents a challenge due to the difficulty of preserving complex 3‐D epitopic sites on the cell surface, the variable expression of antigens on different malignant cell types and the stereotactic interference of closely associated proteins on the intact membrane surface limiting accessibility to antigenic sites. A combinatorial Ig library of 1010 clones was generated from the cDNA of PBMCs derived from patients with breast adenocarcinoma. Following subtractive panning, the library was enriched for Ig (Fab fragment) binding to intact adenocarcinoma cells and the resultant Fabs were screened against a cDNA expression library, itself generated from breast cancer cells. Using this approach, we isolated clones from the cDNA library expressing gC1q‐R, a glycoprotein comprising the major structure of C1, the first component of the complement system. gC1q‐R is a 33 kDa glycoprotein expressed not only on the cell surface but also intracellularly, with motifs that target it to mitochondria and complete homology with HABP and human HeLa cell protein p32, which is copurified with pre‐mRNA SF2. Sequencing of the gene encoding tumor‐associated gC1q‐R did not reveal any consistent tumor‐specific mutations. However, histochemical staining with anti‐gC1q‐R MAb demonstrated marked differential expression of gC1q‐R in thyroid, colon, pancreatic, gastric, esophageal and lung adenocarcinomas compared to their nonmalignant histologic counterparts. In contrast, differential expression was not seen in endometrial, renal and prostate carcinomas. Despite high expression in breast carcinoma, gC1q‐R was also expressed in nonmalignant breast tissue. Although the precise relation of gC1q‐R to carcinogenesis remains unclear, our finding of tumor overexpression and the known multivalent binding of gC1q‐R to not only C1q itself but also a variety of circulating plasma proteins as well as its involvement in cell‐to‐cell interactions suggest that gC1q‐R may have a role in tumor metastases and potentially serve in molecule‐specific targeting of malignant cells.

Butyrate Histone Deacetylase Inhibitors

Abstract In addition to being a part of the metabolic fatty acid fuel cycle, butyrate is also capable of inducing growth arrest in a variety of normal cell types and senescence-like phenotypes in gynecological cancer cells, inhibiting DNA synthesis and cell growth in colonic tumor cell lines, suppressing hTERT mRNA expression and telomerase activity in human prostate cancer cells, and inducing stem cell differentiation and apoptosis by DNA fragmentation. It regulates gene expression by inhibiting histone deacetylases (HDACs), enhances memory recovery and formation in mice, stimulates neurogenesis in the ischemic brain, promotes osteoblast formation, selectively blocks cell replication in transformed cells (compared to healthy cells), and can prevent and treat diet-induced obesity and insulin resistance in mouse models of obesity, as well as stimulate fetal hemoglobin expression in individuals with hematologic diseases such as the thalassemias and sickle-cell disease, in addition to a multitude of other biochemical effects in vivo. However, efforts to exploit the potential of butyrate in the clinical treatment of cancer and other medical disorders are thwarted by its poor pharmacological properties (short half-life and first-pass hepatic clearance) and the multigram doses needed to achieve therapeutic concentrations in vivo. Herein, we review some of the methods used to overcome these difficulties with an emphasis on HDAC inhibition.

Cellular and Molecular Effects of a Pulse Butyrate Regimen and New Inducers of Globin Gene Expression and Hematopoiesis

Abstract: Cooleys anemia is characterized by a deficiency of β‐globin chains, a relative excess of α‐globin chains, and consequent accelerated programmed death of developing erythroid cells in the bone marrow. Increasing expression of the γ‐globin genes to adequately balance excess α‐globin chains can ameliorate this disorder. Butyrates induce γ‐globin experimentally, but can also cause cell growth arrest with prolonged exposure or high concentrations, which in turn can accelerate apoptosis. To determine if these potentially opposing effects can be balanced to enhance therapeutic efficacy, an intermittent “pulsed” regimen of butyrate was evaluated. Following induction of γ‐globin mRNA and protein synthesis, total hemoglobin increased in β‐thalassemia patients by more than 2 g/dl above baseline, and Hb F increased above 20% in 5/8 sickle cell patients from baseline levels of 2% Hb F. Specific regulatory regions were identified in the γ‐ and β‐globin gene promoters to which new binding of transcription factors, including αCP2 (an activator of γ globin) occur during therapy solely in the butyrate‐responsive patients. Other compounds which induce γ globin, derivatives of acetic, phenoxyacetic, propionic, and cinnamic acids, and dimethylbutyrate, are under investigation. Some of these newer γ‐globin inducers (designated hemokines) provide better potential as therapeutics by also acting to increase hematopoietic cell viability and proliferation. Pharmacologic induction of expression of the endogenous γ‐globin genes is a realistic approach to therapy of the β‐globin disorders for many patients, with some effective agents available now and new therapeutics, with enhanced activities, under development.

Identification of novel small-molecule inducers of fetal hemoglobin using pharmacophore and 'PSEUDO' receptor models.

Pharmacologic reinduction of the developmentally silenced fetal (γ) globin genes has been achieved in hemoglobinopathy patients using short chain fatty acid derivatives, with therapeutic effects. However, higher‐potency inducers than are available in currently identified short chain fatty acid derivatives are desirable for long‐term use. Using several short‐chain fatty acids with established γ‐globin induction activity, a pharmacophore template was constructed with the TFIT module of the flo software and used to select several new candidate compounds, three of which exhibited significant activity in a γ‐globin gene reporter transcriptional assay which detects only strong inducers. The data were used to construct a new pharmacophore and a ‘pseudo’ receptor around it. Six hundred and thirty low‐molecular weight compounds were docked into this receptor model. Of 26 compounds selected and tested in functional assays, two compounds showed activity >500% over control levels and two had activity 200% over control range, significantly more active than previously identified short chain fatty acid derivative fetal globin gene inducers. Three compounds had less activity; the remainder showed moderate activity. These findings demonstrate the feasibility of using iterative construction of pharmacophores, pseudo‐binding site modeling, and virtual screening to identify small molecules with the ability to induce transcription of specific target genes, for potential therapeutics.

Induction of Fetal Globin in β-Thalassemia: Cellular Obstacles and Molecular Progress

Abstract: Accelerated apoptosis of erythroid progenitors in β‐thalassemia is a significant barrier to definitive therapy because the beneficial effects of fetal globin‐inducing agents on globin chain balance may not be inducible in cells in which programmed cell death is established early. Accordingly, our objectives have been to identify methods to decrease cellular apoptosis and to identify orally tolerable fetal globin gene inducers. A pilot clinical trial was conducted to determine whether combined use of a fetal globin gene inducer (butyrate) and rhu‐erythropoietin (EPO), the hematopoietic growth factor that prolongs erythroid cell survival and stimulates erythroid proliferation, would produce additive hematologic responses in any thalassemia subjects. Butyrate and EPO were administered in 10 patients. Novel fetal globin gene inducers that also stimulate erythroid proliferation were evaluated for pharmacokinetic profiles. Patients with β+‐thalassemia had relatively low levels of endogenous EPO (<145 mU/mL) and had additive responses to administered EPO and butyrate. Patients with at least one β0‐globin mutation had higher baseline HbF levels (>60%) and EPO levels (>160 mU/mL), and three‐fourths of these subjects responded to the fetal globin gene inducer alone. A few select fetal globin‐inducing short‐chain fatty acid derivatives that stimulated cell proliferation also had favorable pharmacokinetics. These studies identify a significant subset of thalassemia patients who appear to require exogenous EPO to respond optimally to any HbF inducer, as well as new therapeutic candidates that act on both cellular and molecular pathologies of β‐thalassemia. Both approaches now offer excellent potential for tolerable, definitive treatment of β‐thalassemia.

Erythroid Kruppel‐like factor (EKLF) is recruited to the γ‐globin gene promoter as a co‐activator and is required for γ‐globin gene induction by short‐chain fatty acid derivatives

Objectives:  The erythroid Kruppel‐like factor (EKLF) is an essential transcription factor for β‐type globin gene switching, and specifically activates transcription of the adult β‐globin gene promoter. We sought to determine if EKLF is also required for activation of the γ‐globin gene by short‐chain fatty acid (SCFA) derivatives, which are now entering clinical trials.

Evaluation of Safety and Pharmacokinetics of Sodium 2,2 Dimethylbutyrate, a Novel Short Chain Fatty Acid Derivative, in a Phase 1, Double-Blind, Placebo-Controlled, Single-Dose, and Repeat-Dose Studies in Healthy Volunteers

Pharmacologic induction of fetal globin synthesis is an accepted therapeutic strategy for treatment of the beta hemoglobinopathies and thalassemias, as even small increases in hemoglobin F (HbF) levels reduce clinical severity in sickle cell disease (SCD) and reduce anemia in beta thalassemia. Prior generation short chain fatty acid therapeutics, arginine butyrate (AB), and phenylbutyrate, increased fetal and total hemoglobin levels in patients, but were limited by high doses or intravenous (IV) infusion. A fetal globin‐inducing therapeutic with convenient oral dosing would be an advance for these classic molecular diseases. Healthy adult human subjects were treated with a novel short chain fatty acids (SCFA) derivative, sodium 2,2 dimethylbutyrate (SDMB), or placebo, with 1 of 4 single dose levels (2, 5, 10, and 20 mg/kg) or daily doses (5, 10, or 15 mg/kg) over 14 days, and monitored for adverse clinical and laboratory events, drug levels, reticu‐ocytes, and HbF assays. SDMB was well‐tolerated with no clinically significant adverse events related to study medication. The terminal half‐life ranged from 9 to 15 hours. Increases in mean absolute reticulocytes were observed at all dose levels in the 14‐day study. The favorable pharmacokinetics (PK) profiles and safety findings indicate that SDMB warrants further investigation for treatment of anemic subjects with beta hemoglobinopathies.

A randomized phase I/II trial of HQK-1001, an oral fetal globin gene inducer, in β-thalassaemia intermedia and HbE/β-thalassaemia

β‐thalassaemia intermedia (BTI) syndromes cause haemolytic anaemia, ineffective erythropoiesis, and widespread complications. Higher fetal globin expression within genotypes reduces globin imbalance and ameliorates anaemia. Sodium 2,2 dimethylbutyrate (HQK‐1001), an orally bioavailable short‐chain fatty acid derivative, induces γ‐globin expression experimentally and is well‐tolerated in normal subjects. Accordingly, a randomized, blinded, placebo‐controlled, Phase I/II trial was performed in 21 adult BTI patients (14 with HbE/β0 thalassaemia and seven with β+/β0 thalassaemia intermedia, to determine effective doses for fetal globin induction, safety, and tolerability. HQK‐1001 or placebo were administered once daily for 8 weeks at four dose levels (10, 20, 30, or 40 mg/kg per day), and subjects were monitored for laboratory and clinical events. Pharmacokinetic profiles demonstrated a t1/2 of 10–12 h. Adverse events with HQK‐1001 treatment were not significantly different from placebo treatment. The 20 mg/kg treatment doses increased median HbF above baseline levels by 6·6% and 4·4 g/l (P < 0·01) in 8/9 subjects; total haemoglobin (Hb) increased by a mean of 11 g/l in 4/9 subjects. These findings identified a safe oral therapeutic which induces fetal globin in BTI. Further investigation of HQK‐1001 with longer dosing to definitively evaluate its haematological potential appears warranted.

Novel therapeutic candidates, identified by molecular modeling, induce γ-globin gene expression in vivo

The β-hemoglobinopathies and thalassemias are serious genetic blood disorders affecting the β-globin chain of hemoglobin A (α(2)β(Α)(2)). Their clinical severity can be reduced by enhancing expression of fetal hemoglobin (γ-globin), producing HbF (α(2)γ(2,)). In studies reported here, γ-globin induction by 23 novel, structurally-unrelated compounds, which had been predicted through molecular modeling and in silico screening of a 13,000 chemical library, was evaluated in vitro in erythroid progenitors cultured from normal subjects and β-thalassemia patients, and in vivo in transgenic mice or anemic baboons. Four predicted candidates were found to have high potency, with 4- to 8-fold induction of HbF. Two of these compounds have pharmacokinetic profiles favorable for clinical application. These studies thus effectively identified high potency γ-globin inducing candidate therapeutics and validated the utility of in silico molecular modeling.

A phase 2 trial of HQK-1001 in HbE-β thalassemia demonstrates HbF induction and reduced anemia

To the editor: β-thalassemia syndromes comprise a global health burden. Hemoglobin E (HbE)–β thalassemia represents 60% of regional populations, with an increasing prevalence in the coastal United States.[1][1],[2][2] Serious complications related to hemolysis and anemia in β-thalassemia