Chan, Denise A.

Abstract We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

Abstract We and others previously reported that leukemic CLL cells spontaneously secrete VEGF and that VEGF can enhance the leukemic B cell apoptosis resistance via unknown mechanisms. Here, we report elevated VEGF secretion is associated with CLL B cell over-expression of hypoxia inducible factor-1 alpha (HIF-1α), a transcription factor that is a key regulator of VEGF synthesis. Immunostaining analysis confirmed CLL B cells in blood and bone marrow over-expressed both HIF-1α and VEGF. The high HIF-1α levels were not linked to proline residue (402/564) mutations in oxygen dependent degradation domains; absence of proline hydroxylation dependent enzymes (PHD1, 2, 3, and FIH); or to pVHL mutation (exon 1, 2 and 3); or aberrant transcription of HIF-1α. However, pVHL, a regulator of HIF-1α degradation, was found to be lower in CLL B cells and unable to physically interact with HIF-1α in CLL B cells. Nuclear extraction assay revealed that HIF-1α interacted with its co-factor, p300/CRP in B cells, indicating that HIF-1α was transcriptionally active. We hypothesized that reduction of pVHL expression in CLL B cells could be related to repression by micro RNA function which is known to be aberrantly expressed in CLL. We focused on miR-92-1 known to be over-expressed in CLL B cells and it has pVHL as a putative target. In a luciferase assay of transfected human megakaryocytic cell line, MEG-01, we found a direct effect for miR-92-1 on pVHL, with significant repression of luciferase activity of VHL expression by miR-92-1 compared to both control vectors and a mutated target mRNA sequences. This result indicated that wild-type miR-92-1 was able to interact directly with the 3′ UTR sequence of pVHL and subsequently repress pVHL expression. To further confirm this finding, we did transient transfections of either the CLL cell line Mec-1, primary CLL B cells or normal B cells with a sense oligo miR-92-1 (both Mec-1 and CLL B cells) or wild-type miR-92-1 plasmid (normal B cells). We found the level of flow detectable pVHL lower when compared to non-transfected cells. Because we were interested in the relationship of HIF-1α to CLL B cell survival we studied if chemotherapeutic drugs can downregulate HIF-1α in CLL B cells. To do this we studied flavopiridol a drug that has very promising activity in CLL. Our results indicated that flavopiridol does down-regulate HIF-1α levels in CLL B cells. This latter change was associated with induction of apoptosis in CLL B cells. Overall, our data show that elevated VEGF secretion is related to constitutively elevated HIF-1α levels and is an explanation for the VEGF-based autocrine pathway found in CLL B cells. We also show that elevated HIF-1α levels could be partially related to depressed pVHL with a unique mechanism for its repression: the over-expressed microRNA, miR-92-1 in CLL-B cells.

The molecular mechanism of autocrine regulation of vascular endothelial growth factor (VEGF) in chronic lymphocytic leukemia (CLL) B cells is unknown. Here, we report that CLL B cells express constitutive levels of HIF-1α under normoxia. We have examined the status of the von Hippel-Lindau gene product (pVHL) that is responsible for HIF-1α degradation and found it to be at a notably low level in CLL B cells compared with normal B cells. We demonstrate that the microRNA, miR-92-1, overexpressed in CLL B cells, can target the VHL transcript to repress its expression. We found that the stabilized HIF-1α can form an active complex with the transcriptional coactivator p300 and phosphorylated-STAT3 at the VEGF promoter and recruit RNA polymerase II. This is initial evidence that pVHL, without any genetic alteration, can be regulated by microRNA and explains the aberrant autocrine VEGF secretion in CLL.