Von Hippel–Lindau (VHL) small-molecule inhibitor binding increases stability and intracellular levels of VHL protein

Abstract

Von Hippel–Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2VHL). CRL2VHL plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHL:HIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought. Von Hippel–Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2VHL). CRL2VHL plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHL:HIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought. The von Hippel–Lindau (VHL) tumor suppressor is a multisubunit Cullin RING E3 ligase (CRL2VHL)—composed of Cullin2 as the central scaffold subunit, Rbx1 as RING subunit, ElonginB and ElonginC as adaptor subunits, and VHL as substrate recognition subunit (1Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin Jr., W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase.Science. 1999; 284: 657-661Crossref PubMed Scopus (649) Google Scholar, 2Cardote T.A.F. Gadd M.S. Ciulli A. Crystal structure of the Cul2-Rbx1-EloBC-VHL ubiquitin ligase complex.Structure. 2017; 25: 901-911.e903Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). VHL functions to specifically bind hydroxylated hypoxia-inducible factor 1-alpha (HIF-1α), mediating polyubiquitination and subsequent targeting of HIF-1α for proteasomal degradation (3Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4205) Google Scholar). HIFs are transcription factors that regulate the response to reduced oxygen (O2) availability termed hypoxia. HIF is composed of an O2-insensitive β-subunit (HIF-β) that is stable, and an O2-labile α subunit, of which three isoforms are known: HIF-1α, HIF-2α, and HIF-3α. The recognition and ubiquitination of HIF-α subunits by VHL is dependent on proline hydroxylation of the O2-dependent degradation domain of HIF-α, a post-translational modification mediated by 2-oxoglutarate, iron (II) dioxygenases called prolyl-hydroxylases (PHDs). Proline hydroxylation results in a high-affinity binding of HIF-α to VHL and thus subsequent ubiquitination and proteasomal degradation of HIF-α under normal O2 conditions (3Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4205) Google Scholar). Mutations in human VHL result in a number of abnormalities, collectively called VHL disease when occurring in the germ line (4Varshney N. Kebede A.A. Owusu-Dapaah H. Lather J. Kaushik M. Bhullar J.S. A review of von Hippel-Lindau syndrome.J. Kidney Cancer VHL. 2017; 4: 20-29Crossref PubMed Google Scholar). However, VHL is also often loss in clear cell renal cell carcinoma (5Shenoy N. Pagliaro L. Sequential pathogenesis of metastatic VHL mutant clear cell renal cell carcinoma: Putting it together with a translational perspective.Ann. Oncol. 2016; 27: 1685-1695Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The fact that VHL is a tumor suppressor protein, and that VHL loss is found in clear cell renal cell carcinoma tumors, have led to the hypothesis that VHL would not be a good target to inhibit and that chronic HIF stabilization by VHL inhibitors might have detrimental side effects (6Maher E.R. Kaelin Jr., W.G. von Hippel-Lindau disease.Medicine (Baltimore). 1997; 76: 381-391Crossref PubMed Scopus (415) Google Scholar). Apart from HIF, other substrates for VHL have been postulated (7Zhang J. Zhang Q. VHL and hypoxia signaling: Beyond HIF in cancer.Biomedicines. 2018; 6: 35Crossref PubMed Scopus (38) Google Scholar). These include Aurora A, ZHX2, NDRG3, and B-Myb (8Hasanov E. Chen G. Chowdhury P. Weldon J. Ding Z. Jonasch E. Sen S. Walker C.L. Dere R. Ubiquitination and regulation of AURKA identifies a hypoxia-independent E3 ligase activity of VHL.Oncogene. 2017; 36: 3450-3463Crossref PubMed Scopus (26) Google Scholar, 9Zhang J. Wu T. J. M. R. Jonasch E. L. Chen P. M. substrate transcription factor as an in clear cell renal cell 2018; PubMed Scopus Google Scholar, S. M. S.J. H. D.M. response to Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. identifies the protein as a target of the von Hippel-Lindau tumor PubMed Scopus Google Scholar). VHL functions and the of HIF and hypoxia is thus important in a of and conditions T. cellular functions of the von Hippel-Lindau tumor suppressor from PubMed Scopus Google Scholar). We previously to selectively bind to and inhibit VHL J. P. Gadd M.S. L. O. S. M. P. S. Ciulli A. and of hypoxic of HIF-alpha hydroxylation via VHL 2016; PubMed Scopus Google Scholar, P. Gadd M.S. J. L. O. S. Ciulli A. of and inhibitors of the von Hippel-Lindau (VHL) E3 ubiquitin to the 2018; Google Scholar). VH298 is a that the hypoxic response via a different to other HIF that by the VHL:HIF-α of HIF-α hydroxylation J. P. Gadd M.S. L. O. S. M. P. S. Ciulli A. and of hypoxic of HIF-alpha hydroxylation via VHL 2016; PubMed Scopus Google Scholar). We have also characterized the activity of a VH032 Gadd M.S. P. S. S. D.M. Ciulli A. design and of molecules targeting the the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia factor subunit with in PubMed Scopus Google in to induce the response to hypoxia and this to hypoxia or an unbiased J. Ciulli A. S. analysis of and VHL inhibitors reveals and to the hypoxia 4: PubMed Scopus Google Scholar). VH298 also been to the hypoxic response in for in S. J. H. Wu T. von Hippel-Lindau (VHL) protein of and of in by ubiquitination of 2018; PubMed Scopus Google Scholar, S. P. J. G. von Hippel-Lindau (VHL) protein VH298 in by hypoxia-inducible PubMed Scopus Google Scholar). VHL are as of molecules as proteolysis-targeting chimeras M. and PubMed Scopus Google Scholar, Ciulli A. PubMed Scopus Google Scholar, R. D. R.J. the of for protein Full Text Full Text PDF PubMed Scopus Google Scholar). We and have the use of VHL VH032 and VH298 in targeting and M. Ciulli A. induced degradation of the protein PubMed Scopus Google Scholar, J. M. D. J. Chen H. protein degradation as a for S. A. 2016; PubMed Scopus Google Scholar, M.S. A. Chen M. Ciulli A. of recognition for protein 2017; PubMed Scopus Google protein A. E. S. N. N. P. S. G. M. in protein by PubMed Scopus Google Scholar, D. J. G. E. J. to the a 2018; 25: Full Text Full Text PDF PubMed Scopus Google Scholar, H. A. H. N. Ciulli A. D.R. and characterization of an PubMed Scopus Google other target E3 as by VHL S.J. A. Chen S. D.R. R. Ciulli A. of the VHL E3 ubiquitin ligase to induce 2017; PubMed Scopus Google Scholar). the use and of VHL and it is important to the of VHL to the proteome in an unbiased Here, we VHL inhibitors the proteome of quantitative We these to occurring hypoxia or to a IOX2. We that VH032 and VHL protein that is not hypoxia or IOX2 treatment. in VHL protein levels are to stabilization of VHL isoforms and not of VHL protein result in of HIF levels prolonged VH298 treatment in cells. We have unbiased analysis of a broad-spectrum inhibitor and a VHL inhibitor J. Ciulli A. S. analysis of and VHL inhibitors reveals and to the hypoxia 4: PubMed Scopus Google Scholar). we characterized the cellular to a and VHL inhibitor called VH298 J. P. Gadd M.S. L. O. S. M. P. S. Ciulli A. and of hypoxic of HIF-alpha hydroxylation via VHL 2016; PubMed Scopus Google Scholar, P. Gadd M.S. J. L. O. S. Ciulli A. of and inhibitors of the von Hippel-Lindau (VHL) E3 ubiquitin to the 2018; Google Scholar, J. Ciulli A. S. analysis of and VHL inhibitors reveals and to the hypoxia 4: PubMed Scopus Google Scholar). a of the of of VHL by the VHL global proteome analysis in an unbiased a similar to were with with hypoxia and inhibitor by the a of conditions were in with the treatment of a to the global proteome changes induced by HIF and other changes elicited by the VHL inhibitor use of the were with a and We the the by the for protein to the treatment the of in the and this by IOX2 and VH032 and The of for the analysis is which A. J. M. in the is to PubMed Scopus Google Scholar). The for the inhibitors compared with hypoxia be to of inhibitor work is to this of the proteome changes revealed that the of induced by VHL inhibitor were also in the hypoxia and IOX2 and were also in changes to transcription dependent on HIF in the by these HIF-1α is and other are and domain of in and VH032 were a and to for and are with to of these were found in and VH032 by J. Ciulli A. S. analysis of and VHL inhibitors reveals and to the hypoxia 4: PubMed Scopus Google Scholar). in a were a and to for and are with to of these were found in and VH032 by J. Ciulli A. S. analysis of and VHL inhibitors reveals and to the hypoxia 4: PubMed Scopus Google Scholar). hypoxia induced the changes in the induced and protein and This also in the analysis J. Ciulli A. S. analysis of and VHL inhibitors reveals and to the hypoxia 4: PubMed Scopus Google Scholar). HIF such as the VHL inhibitor and to IOX2 resulted in protein with found to be reduced in and This is in line with the that HIF is with and not as a M. L. S. and A on 2018; 6: Scopus Google Scholar). we are in the specificity of the VHL we to the induced by VH032 and VHL. is a protein previously to be induced by hypoxia in S. A. The hypoxia and the of in PubMed Scopus Google not with VHL We to VHL itself. VHL protein upon VHL inhibitor treatment not inhibitor or hypoxia A and to protein of VHL to for the and for the VHL protein in IOX2 for and for and in hypoxia for and for the of the of VHL in the of we VHL protein levels in with VH032 by VHL protein levels not a treatment of in response to a treatment of with VH032 the of the we and characterized a VHL with cell and cellular activity that we as a J. P. Gadd M.S. L. O. S. M. P. S. Ciulli A. and of hypoxic of HIF-alpha hydroxylation via VHL 2016; PubMed Scopus Google Scholar, P. Gadd M.S. J. L. O. S. Ciulli A. of and inhibitors of the von Hippel-Lindau (VHL) E3 ubiquitin to the 2018; Google Scholar). VH298 the binding HIF-1α and as in we VH298 in VH298 treatment resulted in in VHL protein levels also in a that this is to the of VHL by the molecules. the in VHL protein levels not with the and that of VHL results specifically from binding to VHL. VH298 also VHL protein levels in cell human and the treatment of hypoxia or inhibitor IOX2 not VHL protein levels and in with the proteomic results A and We also that treatment with which is a similar results to with in VHL protein levels. VHL protein levels were to be by the VHL not in in which the proteomic analysis also in and this not hypoxia or in the of We the in VHL protein levels in the of VHL inhibitor to the in levels. quantitative VHL levels of VHL were not in the of VH032 or VH298 in or VH298 in to the VHL protein hypoxia and IOX2 not induce changes in VHL levels We VHL might be to be by the similar to VHL the proteasomal degradation of However, results that VHL protein levels not in the of inhibitor We the VHL might be the We a that protein and VHL protein levels to the of VHL in the of VHL revealed of the of VHL and the and VHL VHL a and a O. A. S. Kaelin Jr., W.G. by the human von Hippel-Lindau PubMed Scopus Google Scholar, O. M. Kaelin Jr., W.G. is a of the von Hippel-Lindau from S. A. PubMed Scopus Google Scholar). These isoforms are also as and on their upon protein from an from the of VHL and the domain O. M. Kaelin Jr., W.G. is a of the von Hippel-Lindau from S. A. PubMed Scopus Google Scholar, A. A von Hippel-Lindau from an functions as a tumor S. A. PubMed Scopus Google Scholar). The VHL inhibitor VH298 the of from to the of to the treatment. This result to the protein levels for and in the of VHL inhibitors and these that the binding of VHL inhibitor to VHL protein as a result of protein and degradation. We have previously that HIF-1α protein levels are in the of VH298 of of the VHL:HIF-α interaction, this is not in prolonged treatment and J. P. Gadd M.S. L. O. S. M. P. S. Ciulli A. and of hypoxic of HIF-alpha hydroxylation via VHL 2016; PubMed Scopus Google Scholar). VHL protein levels in the of we that the of HIF-1α levels in prolonged treatment of VHL inhibitor be to the of VHL protein levels. we we the HIF-1α by VH298 to inhibit the VHL. We the of VH298 to induce the of HIF-1α, and the were found to be and in and The of VH298 to that been with VH298 for which HIF-1α protein levels would have HIF-1α protein levels were the subsequent of VH298 HIF-1α the of VH298 not the HIF-1α to the as the HIF-1α in and cells. These results that the of HIF-1α levels in prolonged VH298 treatment not of VH298 to inhibit VHL. However, it is also that the inhibitor may be by cell and may be to the VHL levels under we the HIF-1α is in a by of VHL in by VH298 treatment. VH298 treatment resulted in of HIF-1α as under VHL by HIF-1α the of treatment This that the degradation of HIF-1α mediated by VHL. Interestingly, of VHL led to stabilization of HIF-1α and as previously S.J. A. Chen S. D.R. R. Ciulli A. of the VHL E3 ubiquitin ligase to induce 2017; PubMed Scopus Google Scholar). This is with not to and that of the VHL to be to HIF-1α for proteasomal degradation. We that the activity and of VHL might the of of VHL inhibitor in to levels of this a VH298 treatment with and VHL the of VHL HIF-1α levels be of in to the HIF-1α levels were the of VH298 of VH298 a inhibitor treatment under VHL found to similar HIF-1α stabilization as VH298 in the The of VH298 and the of VHL on HIF-1α these that treatment with VHL inhibitor HIF-1α is and in a as VHL HIF-1α is to be via the proteasomal S. of by J. PubMed Scopus Google as as J.S. P. H. ubiquitin is a for degradation by PubMed Scopus Google the in the degradation of HIF-1α, were with VH298 for by inhibitor or inhibitors or for the HIF-1α upon of the three inhibitors of VH298 the HIF-1α to similar protein levels as treatment of VH298 inhibitors were not as and HIF-1α to a compared with these that in prolonged VH298 the HIF-1α that in the of VHL inhibitor VH298 are via the proteasomal in a this we the of a VHL inhibitor VH032 the proteomic quantitative MS VH032 effects were compared with elicited by hypoxia and the broad-spectrum inhibitor IOX2. This analysis analysis J. Ciulli A. S. analysis of and VHL inhibitors reveals and to the hypoxia 4: PubMed Scopus Google as that we were also the This is as global have reported a of to protein S. R. analysis of and proteomic PubMed Scopus Google Scholar). are induced different of the hypoxia response, and protein and might also be in hypoxia. we to the VHL inhibitor would have effects the protein not the Our analysis that HIF an of that are The of as induced in analysis with as from the HIF-1α and HIF were also This that HIF are induced different in the hypoxia response and that a analysis of and protein would be to induced by the VHL been to activity G. H. The of factor in renal cell J. PubMed Scopus Google Scholar, Q. L. H. M. J. ubiquitination of to 2016; PubMed Scopus Google Scholar, D. E. M. R. J. L. E. R. M. M. VHL response renal cell carcinoma via regulation of 2017; PubMed Scopus Google VH032 found to target the HIF response, as in proteomic or in J. Ciulli A. S. analysis of and VHL inhibitors reveals and to the hypoxia 4: PubMed Scopus Google Scholar). This the specificity of the VHL inhibitor in this the use of VHL inhibitors and as to and as we were in effects and identify Our analysis revealed that VH032 is and for VHL. the protein by not previously to be induced by VHL as previously been as a hypoxia-inducible protein S. A. The hypoxia and the of in PubMed Scopus Google Scholar). of VHL also by the inhibitor analysis revealed that this is dependent on VHL stabilization and not in levels. We have previously binding of VH298 to which in resulted in on the protein A. S. Ciulli A. the von Hippel-Lindau factor Full Text Full Text PDF PubMed Scopus Google as as in a cellular J. P. Gadd M.S. L. O. S. M. P. S. Ciulli A. and of hypoxic of HIF-alpha hydroxylation via VHL 2016; PubMed Scopus Google Scholar). Interestingly, results suggest that levels of VHL are for reduced HIF-1α levels prolonged to Our suggest that the levels of VHL is to the of are for these from a it that the HIF VHL protein as in to levels of the HIF transcription factor for prolonged This to the of and protein levels A. N. J. E. chronic hypoxia and from S. A. PubMed Scopus Google Scholar). it that changes to VHL levels can in regulate HIF-1α levels in cells. This is a that proline hydroxylation is in to HIF-1α levels (3Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science. 2001; 292: 468-472Crossref PubMed Scopus (4205) Google VHL levels can an to HIF-1α levels this remains and is to this Our are also to the of VHL inhibitors such as VH298 or as as This is important the use of these compounds as to and the in their therapeutic VH298 been to S. P. J. G. von Hippel-Lindau (VHL) protein VH298 in by hypoxia-inducible PubMed Scopus Google and also S. J. H. Wu T. von Hippel-Lindau (VHL) protein of and of in by ubiquitination of 2018; PubMed Scopus Google in a of be anemia and mitochondrial HIF stabilization been to be of S. T. M. factor for anemia of chronic 2018; 27: PubMed Scopus Google Scholar, L. R. S. H. O. J. O. Zhang as a for mitochondrial 2016; PubMed Scopus Google Scholar). Our results target specificity of VH298 and to reduced HIF-1α levels upon prolonged inhibitor treatment suggest that the use of a of this in these conditions be as it would be to have side effects in However, analysis is in in these conditions to this to to to reported of VHL levels upon treatment. This may be in of the fact that VHL protein levels not to be with the of and E3 ligase degradation S.J. A. Chen S. D.R. R. Ciulli A. of the VHL E3 ubiquitin ligase to induce 2017; PubMed Scopus Google Scholar, M. S.J. A. Ciulli A. versus E3 other 27: PubMed Scopus Google Scholar). is also to that VHL stabilization not be of the of of to induce target protein as to the of of inhibitors M. and PubMed Scopus Google Scholar). we for the to protein levels of VHL when Interestingly, of the E3 ligase VHL in been to and the reduced of S. Zhang D. Zhang Q. Zhang P. D. J.S. J. Zhang A. and 25: PubMed Scopus Google Scholar). We a a inhibitor can the levels of target protein, to a in substrate recognition and enzyme this may not be we were to other such in the the enzyme which is induced upon stabilization by inhibitor Z. of occurs by enzyme for of 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). or enzyme target is often to a in or which in turn the transcription the of the for the enzyme target A. C.L. of and activity of S. A. PubMed Scopus Google Scholar, M. M. M. P. regulation of target PubMed Scopus Google Scholar). The result is an in target protein, to of target as a result of P. of A Oncol. PubMed Scopus Google or of the enzyme from binding to P. D. S. M. M. T. J. R. S. J. R. to cellular levels of to A to S. A. PubMed Scopus Google Scholar). Here, results and that protein stabilization is the for the levels of VHL. The that VHL inhibitors their target protein is with of VHL structure and is that VHL as a tumor suppressor (6Maher E.R. Kaelin Jr., W.G. von Hippel-Lindau disease.Medicine (Baltimore). 1997; 76: 381-391Crossref PubMed Scopus (415) Google and that the VHL protein J. P. Gadd M.S. L. O. S. M. P. S. Ciulli A. and of hypoxic of HIF-alpha hydroxylation via VHL 2016; PubMed Scopus Google Scholar, A. S. Ciulli A. the von Hippel-Lindau factor Full Text Full Text PDF PubMed Scopus Google and cellular We that VHL stabilization upon binding to a in degradation of VHL. We that such from degradation be the result of or by to other cellular or by and on the VHL protein that the by which VHL inhibitors VHL in the this work demonstrates the specificity of VHL inhibitors VH032 and VH298 and that these inhibitors levels of their target protein as a result of protein binding and This important for the of VHL inhibitors and We suggest that chronic use of VHL inhibitors might from VHL and VHL might not be as detrimental to or as previously thought. carcinoma and were from and in with and of were for from hypoxia were O2 in an hypoxia to were for protein in the hypoxia as The inhibitor from and the of for inhibitors IOX2 and were from VHL inhibitors and were by a of J. P. Gadd M.S. L. O. S. M. P. S. Ciulli A. and of hypoxic of HIF-alpha hydroxylation via VHL 2016; PubMed Scopus Google Scholar, P. Gadd M.S. J. L. O. S. Ciulli A. of and inhibitors of the von Hippel-Lindau (VHL) E3 ubiquitin to the 2018; Google Scholar). were to for of and were to for and were from and in a of were from to the The for are the were in and a inhibitor were in a and with as in cells. were for is and with a for were in as of cell were were with of HIF-1α or of in a of were to the by in a for were with The were from with and as previously by the to the in in levels were on and to levels. are were with hypoxia and VH032 for were in in and inhibitor of were for of under with and for be The were in The protein were and to the previously M.S. A. Chen M. Ciulli A. of recognition for protein 2017; PubMed Scopus Google Scholar). of conditions of as a of in the MS that not of this The treatment VHL inhibitor which led to the results of is not P. Gadd M.S. J. L. O. S. Ciulli A. of and inhibitors of the von Hippel-Lindau (VHL) E3 ubiquitin to the 2018; Google Scholar). were to that in of on a with a The from were a of A and in and and in The were from the a of with a from A to in and to by The with for and in A for The in A from to with a of a of and an of by an to and the on the that the are MS from were human the to as the enzyme to The the and The were by a were the conditions to of the for and for the findings of this are the and from the upon The MS have been to the via the with the This The Ciulli or from and A. is a and of a that is protein degradation therapeutic The other that have of with the of this We and from the for in and analysis of MS for with MS and for J. S. and A. J. S. and A. J. and A. J. and A. J. S. and A. J. and A. J. and A. J. S. and A. J. and A. J. S. and A. J. S. and A. J. S. and A. J. S. and A. and This work by the a to J. the a to A. and Cancer a to S. R.

Topics & Concepts