Pantothenate kinase isoforms as collateral lethality targets in Glioblastoma Multiforme NAME OF STUDENT On-Topic Candidacy Exam Adviser: Dr. XXXXXX
Concomitant l loss o of p passeng nger ge genes es o occur with genomic c de deletion of tum umor s r supp uppressor g r genes TCGA Pan-Cancer Dataset DNA Copy Number =0 =1 =2 >2 PTEN Coenzyme A Biosynthesis PANK1 1
Prev evalence o e of PT PTEN-PANK1 homozygous d deletions a across c cancer er t types es PANK1 homozygous deletions are found in cancers with PTEN homozygous deletions PTEN • Prostate Adenocarcinoma • Glioblastoma • Ovarian Cancer • Melanoma PANK1 PANK1 • Cervical Carcinoma 2
Pantothenate kinases s ar are r rate l limit itin ing f fac actors i in coenzyme e A A production O O OH HO N Pantothenate (vitamin B 5 ) Pantothenate H OH PANK O O O 4-phosphopantothenate O P OH HO N H O OH 4-phosphopantothenoyl L-cysteine Pantothiene-4’-phosphate O O O HS O P O N N Dephospho-CoA H H OH O O HO O P O HO O P O O O Coenzyme A (CoASH, CoA) N N N N NH 2 Extracellular Intracellular 3 Plasma membrane
Coenzyme e A A regulates es a a multit itude of of e essentia ial c l cellu llula lar f functio ions Food intake Vitamin B 5 Regulation Acyl group Carrier Coenzyme A Lipid Metabolism Fatty Acid oxidation and Acetyl CoA Protein acetylation Biosynthesis TCA Cycle CO 2 + Energy 4
PANK ANK i isoforms h have e distinct c cellular l localization and nd tissue s e spec ecific distribution • PANK1 PANK1 ⍺ (nuclear; Liver and kidneys) PANK1β (cytosolic; Liver) • PANK2 (mitochondrial; neuronal tissue) • PANK3 (cytosolic; all tissue types) • PANK4 (catalytically inactive) Alfonso-Pecchio A et al (2012) PLOS One, 7: 11, Figure 2 5
Cell e essen entiality o of P PANK ANK i is ev eviden enced b by PANK ANK knockout phen enotype e in n lower er o organisms • E. coli , S. cerevisiae and D. melanogaster only have one PANK isoform • PANK knockout is lethal in all unicellular organisms 6
Organismal e essentiality o of PANK ANK is ev eviden enced b by PANK ANK isoform k knockout p phen enotype pes i in mi mice • Individual PANK paralog knockout mice are viable • Pank1 -/- : Hepatic fatty acid oxidation and gluconeogenesis impaired in fasted state • Pank2 -/-: Pantothenate Kinase Associated Neurodegeneration in human; retinal degeneration and impaired spermatogenesis in mice • Pank3 -/- : No known phenotype • Double knockout mice are embryonic or post natal lethal • Pank1 -/- Pank3 -/- and Pank2 -/- Pank3 -/- dko mice are embryonic lethal • Pank1 -/- Pank2 -/- dko are late post-natal lethal Conclusion: At least two PANK isoforms are required for organism viability 7
CENT NTRAL HY RAL HYPOTHESIS IS Targeting the redundant isoforms of PANK in tumors with PANK1 homozygous deletion can selectively kill cancer cells. Normal tissue Tumors with PANK1-/- Vitamin B5 / Vitamin B5 / Pantothenate Pantothenate PANK2/3 PANK1 PANK1 PANK2/3 Co Enzyme A Co Enzyme A Vitamin B5 / Vitamin B5 / Pantothenate Pantothenate shPanK2/3 shPanK2/3 PANK1 PANKK2/3 PANK1 PANK2/3 Co Enzyme A Cell death 8
Speci cific c Aim m 1 To determine if PANK activity is cell essential and identify the paralog redundant with PANK1 in cancer cells Hypothesis : Based on the cytosolic co- localization of PANK1 β and PANK3, I hypothesize that PANK3 is redundant with PANK1 β and therefore compensates for loss of PANK1 . 9
Speci cific A c Aim 1 To determine if PANK activity is cell essential and identify the paralog redundant with PANK1 in cancer cells Identification and generation of cancer cell lines with PANK1 homozygous deletion • Test the effect of a pan-PANK inhibitor and antisense oligonucleotides against PANK • isoforms CRISPR KO of PANK isoforms alone or in the context of PANK1 homozygous deletion • Generation of PANK1 isogenic rescued cell lines • • Constitutive and inducible knock down of PANK2 and PANK3 in PANK1 deleted and intact cells Cell viability assays • 10
AVANA C ANA CRISPR d R data s set et i identifies P PANK ANK isoforms as non-essen ential ge genes es PANK1 (53354) PANK2 (80025) PANK3 (79646) PANK4 (55229) 11
Selec ection o of in v vitro model el w with PANK ANK1 homozygous d s deletion N=676 PANK1 50 TPI (Loading control) 25 Background 12 Analyzed based on data from : Klijn, C et al. 2015, Nature Biotechnology 33: 306–312, Supplemental Data 1
Gener eration o of PANK ANK1 CRISPR R KO cancer c cell l lines es PANK1 50 50 PANK1 37 TPI 27 TPI 25 Gastric Sarcoma Breast Conclusion: PANK1 is dispensable in cancer cells 50 PANK1 37 TPI 25 13
Gener eration o of PANK ANK1 isogen enic rescued ed c cell l lines Lentiviral pCMV- vectors + PANK1 psPAX2 , pMD2.G Expected Western Blot result Transfection 293T cells PANK1 TPI (Loading control) G59 PANK1 OE cells G59 cells Puromycin selection PANK1 over expression 14
Gener eration o of He HeLa La PANK ANK2 and nd PANK ANK3 CRISPR K R KO line Anti-PANK2 and anti-PANK3 antibody development in progress 15
Expected outcome: PANK isoforms are individually dispensable PANK isoforms protein level in HeLa cells PANK1 PANK2 PANK3 TPI (Loading control) 16
PANK ANK1 homozygously deleted ed 537 37-MEL cel cells respond to to PANK ANK3 inhib ibit itio ion 25 12.5 3.15 6.25 0 ASO (µM) 537-MEL PANK1 -/- SK-MEL5 PANK1 +/+ SK-MEL-28 PANK1 +/+ Day 4 17
Compound 7 7, a a Pan-PAN ANK i inhibitor has modest s t selecti tivity ty for P PANK3 K3 PANK Isoforms IC50 PANK1 β 70 ± 1.1 nM PANK2 92 ± 2.0 nM PANK3 25 ± 1.8 nM 18 Sharma et al J Med Chem. 2015 Feb 12; 58(3): 1563–1568.
Compound 7 7 shows s selec ective e toxicity t to P PANK ANK1 1 deleted ed me melanoma t tumo mor-spheres es Compound 7 PANK inhibitor 0 uM 3.12 uM 6.25uM 12.5 uM 25uM 50uM 537-MEL PANK1 -/- SK-MEL5 PANK1 +/+ SK-MEL-28 PANK1 +/+ Day 6
shRN RNA m A mediated k knockdown of PANK ANK2 2 and PANK ANK3 in n G5 G59 c cells ls Expected Result 10 individual short hairpins 2nd G59 and G59 PANK1 OE cells PANK1 generation ( Puromycin selection) pLKo- lentiviral PANK2 shPANK + Identify shRNA clones with KD>60% packaging TPI 2/3 To clone into inducible system pTRIPZ vector psPAX2 , pMD2.G PANK1 PANK3 TPI 20
Ex Examin inin ing t the e effect of of l los oss of of PANK ac activ ivit ity on on c can ancer c cell v ll viab abilit ility • Cell viability Assay with Crystal violet staining/Cell-titre-glo assay G59 shPANK2/3 cells • Colony formation Assay • Cell growth Analysis by IncuCyte • FACS with annexin V-PE and 7-AAD G59 shPANK2/3 pCMV to detect apoptosis PANK1 cells 21
Expect cted R Result: P : PANK3 inhibition will select ctively y imp mpede PANK1 nul null tum umor ce cell growth in n vitro G59 PANK1 -/- G59 PANK1 OE *** Dox - + - + - + Dox - + - + - + ishScr ishPANK3 ishPANK2 ishScr ishPANK3 ishPANK2 • Slow rate of cell growth in G59 shPANK3 • Increased annexin V-PE and and reduced 7-AAD 22
AIM1- Pitfal alls ls/Alternative Approac oach Individual isoform may be essential and CRISPR KO may not be feasible. • Recently released AVANA dependency map identifies PANK isoforms as non essential shRNA mediated knock down of PANK may lead to a complete cell death • Modulation of dox concentration to measure the effect of acute loss of PANK activity. If shRNA mediated knock down is not informative in identification of the redundant isoform Generate inducible CRISPR KO of PANK isoform. • If PANK activity is dispensable in cancer cells, it will contradict my hypothesis, but also suggest that cancer cells can survive without Co-enzyme A. • Unlikely, because all other downstream enzymes in the CoA biosynthesis pathway are essential based on the dependency score, suggesting that this pathway is essential. 23
Spe pecifi fic Ai Aim 2 To determine the biochemical consequences of PANK ablation Hypothesis : I hypothesize that the loss of PANK activity will deplete CoA level from the cells which can impact critical metabolic and transcriptional profile of the cells. 24
Specific Ai Aim 2 2 To determine the biochemical consequences of PANK ablation Profile small molecule metabolites by metabolomics • Determine the effects of protein acetylation • • Identify key signaling pathways by RPPA • Transcriptomics following PANK ablation in PANK1 homozygously deleted and PANK1 intact cells.
Co Co-enzyme A A pl plays c critical r role i in n a mul ultitude o of f bi biochemical r reactions AcetylCoA is required for all protein acetylation reaction! 26
Met etabolomics to s to iden enti tify k key ey m met etabolites a es alter tered ed b by co-en enzyme A e A dep epleti tion G59 shPANK3 cells Mass spectrometry Extraction of polar metabolites with (LC/MS) 80% methanol at -80 C ASARA MS core at BIDMC G59 PANK1 OE shPANK3 Expected Outcome: Analysis of ther PANK1 intact Glioma Cells: Massive disruption of Fatty Acid • metabolites D423, D502, LN319 Biosynthesis and oxidation pathway Decreased metabolites in the CoA • synthesis pathway Decrease in OCR and increase in • glycolytic intermediates, as well as ECAR 27
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