Pantothenate kinase isoforms as collateral lethality targets in - PowerPoint PPT Presentation

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