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Steven R. Ellis, PhD Research Directo

This month's Journal Club is, in part, a literary piece including a biography of one of the authors of the manuscript under consideration.  This biography will be in the Hellenist tradition, focusing on big picture items, rather than a detailed chronology of a person's life.  Is this biography of one of the elder statesmen (or women) in the DBA field?  Actually not, this person is in his late 20's early 30's max, but has already accomplished much at this young age.  The subject of this biography is Dr. Johan Flygare, physician/scientist by choice, Swede by birth.  When I entered the DBA field in the mid 2000's, Johan was already making a name for himself as a graduate student in Dr. Stefan Karlsson's lab at Lund University in Sweden.  Their group had created one of the more sophisticated cellular models of DBA.  During this period, Johan also spent a brief period in my laboratory and together with Dr. Anna Aspeci, who was visiting from Irma Dianzani's lab in Italy, we were able to show a ribosome biogenesis defect in cells from DBA patients.  In the late 2000's Johan had to return to clinical training, but after receiving his MD he returned to the Karlsson lab briefly before entering the laboratory of Dr. Harvey Lodish at the Whitehead Institute in Boston.  It is a manuscript stemming from his time in the Lodish lab that is the subject of this Journal Club.  This manuscript has substantial implications for the development of improved therapies for Diamond Blackfan anemia.

The manuscript (Flygare et al) published in Blood (2011) 117:3435-3444 is entitled "HIF1α synergizes with glucocorticoids to promote BFU-E progenitor self-renewal".  As you are all aware, steroids are a therapeutic option for DBA, but come with a number of potential complications. The work by Flygare and colleagues shows that drugs that increase the amount of the protein HIF1α work together with glucocorticoids to stimulate the production of erythorid progenitor cells found in reduced amounts in DBA patients.  These studies suggest that it might be possible someday to treat DBA with reduced steroid doses, or eliminate exogenous steroids altogether using drugs that increase HIF1α levels.  Much still remains to be worked out before this approach will translate to the clinic, but it is encouraging to note that drugs targeting HIF prolyl hydroxylases are currently being considered as alternative treatments for EPO-responsive anemias so their toxicity profiles and other pharmacological properties are currently being examined (Muchnik & Kaplan).

Let us turn our attention back to the Flygare manuscript.  The study begins by describing a new technique to isolate the hematopoietic progenitor cell populations thought to be primarily affected in DBA.  They were then able to show that it is the BFU-E progenitor cells that are responsive to glucorticoids, stimulating their self renewal and thereby increasing the amount of erythroblasts that can be derived from individual BFU-Es approximately 40-fold.  To understand how glucocorticoids were having this effect, Flygare and colleagues examined changes in gene expression induced by glucocorticoids.  Somewhat surprisingly, the authors found that many of the genes upregulated by glucocorticoids contained DNA sequence signatures characteristic of genes regulated by the protein HIF1α.  HIF1α regulates gene expression in response to low oxygen concentrations.  A role for HIF1α in erythropoiesis has been known for some time as a regulator of erythropoietin (EPO), which makes a great deal of sense, because if oxygen concentrations are low, a reasonable response would be to induce erythropoietin expression and make more red blood cells.  HIF1α itself is regulated by a family of enzymes known as HIF prolyl hydroxylases, which use molecular oxygen to label the HIF1α protein for degradation.  When oxygen partial pressures are low, the HIF prolyl hydroxylases targeting HIF1α are inhibited, leading to higher levels of HIF1α which in turn stimulate erythropoietin production.  The surprise in the Flygare manuscript however, was that HIF1α also appears to be involved in inducing erythropoiesis at an earlier stage upstream of EPO, acting on BFU-E's to stimulate their self-renewal.

The authors reasoned that by inhibiting the HIF prolyl hydroxylases targeting HIF1α they could potentially increase HIF1α levels and stimulate erythropoiesis at the progenitor level.  Moreover, they felt that glucocorticoids and prolyl hydroxylase inhibitors might work together or at least show some partial overlap in stimulating erythropoiesis at the progenitor stage.  When put to the test, they were able to show that a drug targeting HIF prolyl hydroxylases enhanced the effect of glucocorticoids in stimulating BFU-E's by about 7 fold.  Thus, the two drugs have a synergistic effect in inducing the production of CFU-E's and erythroblasts from BFU-E's. 

What has all this to do with DBA?  Plenty, in fact.  To quote the authors, "A group of patients who directly could benefit from the CFU-E-promoting effect of PHIs [Prolyl Hydroxylase Inhibitors] are those with the red cell progenitor disorder DBA."  The results from Flygare et al. suggest that with the synergistic effect seen with prolyl hydroxylase inhibitors it may be possible to reduce steroid concentrations in treating DBA patients or possibly eliminate exogenous steroids altogether through the ability of prolyl hydroxylase inhibitors to synergize with endogenous levels of steroids found in DBA patients. 

So is there a downside to all of this?  Well, yes; inhibiting prolyl hydroxylases that target HIF1α for degradation could lead to an enhanced risk of cancer.  Once marked for degradation by prolyl hydroxylases, another protein is necessary to direct the actual degradation of HIF1α.  A genetic deficiency of this other protein leads to von Hippel-Lindau syndrome, a strong cancer predisposition syndrome.  The rationale for this increased incidence of cancer is in part, that tumors are frequently hypoxic (reduced oxygen supply) and need HIF1α expression to counteract the hypoxia.  Thus, promoting HIF1α expression by using prolyl hydroxylase inhibitors could in principle lead to an increased risk for cancer.  While there is certainly cause for proceeding with caution on the potential use of prolyl hydroxylase inhibitors as a treatment option for DBA, these studies nevertheless open a new window of opportunity for improved treatment options for patients with DBA.

One final note in this rather lengthy journal club is that Dr. Flygare has been supported by the DBA Foundation at various points in his career.  He is transitioning from the Lodish lab to begin his own laboratory at the same Institute as that of Stefan Karlsson in Lund Sweden.  One of the proposals currently under review for funding from the DBA Foundation is from Dr. Flygare as he starts this new phase of an extremely productive career.                            
Flygare J, Rayon Estrada V, Shin C, Gupta S, Lodish HF HIF1alpha synergizes with glucocorticoids to promote BFU-E progenitor self-renewal. Blood 117(12): 3435-3444

Muchnik E, Kaplan J HIF prolyl hydroxylase inhibitors for anemia. Expert Opin Investig Drugs 20(5): 645-656