DNA repair, oncometabolites and cancer therapy

Transcription

1 DNA repair, oncometabolites and cancer therapy Peter M. Glazer, M.D., Ph.D. Robert E. Hunter Professor and Chair, Department of Therapeutic Radiology Professor of Genetics Yale School of Medicine

2 Disclosures I have nothing to disclose.

3 Actual conversation at a Cold Spring Harbor meeting, 2017 I work on DNA repair I used to ignore DNA repair, but I'm beginning to think it's important. Doug Brash Professor of Therapeutic Radiology Yale School of Medicine James Watson

4 Mutations per Mb Genomic instability is a hallmark of cancer Somatic Mutations Chromosomal aberrations

5 Identification of frequent mutations in Isocitrate dehydrogenase genes (IDH1 and IDH2) in gliomas Normal Function of IDH1/2: Covert isocitrate to alpha-ketoglutarate (AKG) in the Kreb s cycle (Citric acid cycle) Gene IDH1 IDH2 Function Alteration Frequency Citric Acid Cycle 78% Citric Acid Cycle 5%

6 IDH1, Isocitrate Dehydrogenase 1, participates in the Citric Acid Cycle The same mutation in almost all cases 80% of Low Grade Glioma 20% of Cholangiocarcinomas 19% of AML 6% of Melanomas 5% Chondrosarcomas

7 Neomorphic IDH mutants produce 2-Hydroxyglutarate (2HG) Neomorphic IDH1 Converts AKG to 2HG 2HG is produced at 5-10 mm in midh Gliomas

8 Hypothesized oncogenic mechanism: 2HG competes with AKG to inhibit AKG-dependent dioxygenases that regulate gene expression Alpha-Ketoglutarate AKG- Dependent Dioxygenase s 2-Hydroxyglutarate (2HG) TET proteins: DNA Methylation KDM4C: Histone Demethylation

9 IDH1/2 mutations in glioma: Friend or foe? Strategy to develop small molecules to inhibit the neomorphic IDH activity Anaplastic Astrocytoma Convert to WT IDH1???

10 Screen reveals that IDH1 mutations confer exquisite PARP inhibitor sensitivity Ranjit Bindra Two key questions emerge: 1. Is this a defect in homologous recombination (i.e., BRCAness?) 2. Is the HR defect mediated by the oncometabolite, 2HG?

11 γh2ax-positive cells et Tail Moment eactivation by NHEJ eactivation by NHEJ Comet Tail Moment +Dox Mut Mut -Dox Norm. 2HG Levels WT WT Surviving Fraction Surviving Fraction Cell Viability (96 h) Interrogating the functional consequences of IDH1/2 mutations Evidence for a DNA repair defect Comet Assay 101 Increased comet tail moment correlates with DSB repair defects Neutral Comet Assay quantifies DNA Double strand break persistence Single Cell DNA electrophoresis DNA in tail is double strand breaks Example: BRCA2 mutant cells harbor an HR defect with comet tails Supplementary Figure S2 A D 40 (R132H IDH1) THP1 Cells HCT116 WT R132H/ IR Dose (Gy) B HeLa WT R132H/ IR Dose (Gy) 3K THP1 Cells E C HEL Cells (Post-IR; 5 Gy ) Parker 0.2 Sulkowski Grad 0.1 student Glazer lab Dox WT R132H F HeLa Cells 20 2K IDH1/2-mutant cells show increased comet tails similar to BRCA2-mutant cells Comet tails G Dox WT R132H (IDH1) 1K Dox WT R132H (IDH1) H I DNA damage response foci γh2ax J IDH1 mutant cells show elevated 20 gh2ax and 53BP foci 10

12 IDH1 mutant cells show radiation sensitivity which also points to DNA repair defect Sulkowski et al Science Translational Medicine

13 IDH1 mutant cells have a persistence of DNA double strand breaks post IR Sulkowski et al Science Translational Medicine

14 Cells with IDH1 or IDH2 mutations have deficiency in homologous recombination Sulkowski et al Science Translational Medicine HEL cells engineered to have inducible expression of genes with IDH1 R132H or IDH2 R172K

15 ized HR Activity Cells (HR Activity) et Tail Moment et Tail Moment HR Capacity Normalized Cell Kill %GFP Postive Cells %GFP Postive Cells HR Efficiency %GFP Postive Cells Mean Mean Comet Comet Tail Moment Tail Moment (R)-2HG sicon sirad51 %GFP+ Cells (HR Activity) Unresolved DNA DSBs by Comet %GFP+ Cells (HR) Mean Comet Tail Moment Robust Z-Score Mean HR Comet (DR-GFP) Tail Moment sibrca2 sixrcc4 Mean Comet Tail Moment (R)-2HG Relative HR Capacity Fractional Cell Number Mean Comet Tail Mo 0 Mean Comet Tail M Mean Comet Tail Mo %G Dose-dependent suppression of homologous recombination 300 by YUKIM YURL Concentration (um) Figure (R)-2HG (um) 3 2HG and F DMG in <24 h G R2HG R2HG S2HG R2HG S2HG DMG D2HG-Acid S2HG DMG D2HG-Acid DMG AKG D2HG-Acid AKG AKG HG DMG AKG I M (2S)-ctyl-α-hydroxyglutarate p= (R)-2HG (µm) J BRCA2 150Actin Vinculin 0 SCR TARGET (2R)-ctyl-α-hydroxyglutarate 100 RAD Vinculin 50 XRCC4 0.0 DMG (2S)-ctyl-α-hydroxyglutarate um 2HG HeLa 2 2 DLD1 300uM 2HG BRCA2 Loss PE Concentration (um) sirnas HCT116 Cells DMG nm BMN YUKIM Concentration (um) (R)-2HG (um) KMD4A Si1 Si2 Si3 A F N H H H N H H p= Concentration (mm) Mock siscr Si1 Si2 Si3 HCT116 B K H Immortalized Astrocytes1.0 0 um 30 um 2HG um 300 um DMG H N H 1 BMN- ctylα-kg Concentration (mm) BRCA2 Actin RAD51 SCR -R C 0.5 TARGET 0 HCT116 + HCT116 + Primary Melan L YUKIM G-5 WT B BRC DM

16 2HG induced HR suppression is on par with BRCA loss Sulkowski et al Science Translational Medicine

17 2HG is necessary and sufficient for the IDH1 R132H dependent DSB repair defect Sulkowski et al Science Translational Medicine

18 Exogenous KG can rescue the DSB repair defect -Ketoglutarate 2HG R132H/+ HeLa AKG-Dependent Dioxygenases

19 But which KG-dependent dioxygenase?

20 Sulkowski et al Science Translational Medicine Finding the target sirna Screen of AKG Dependent Dioxygenases

21 Forced over-expression of KDM4A and KDM4B can rescue the comet phenotype in IDH1 R132H mutant cells Conclusion: Inhibition of KDM4A/B mediates 2HG-induced HR suppression ther KG-dependent dioxygenase RFs do not rescue Sulkowski et al Science Translational Medicine

22 Is this relevant for gliomas? 2HG exposure is sufficient to confer PARPi sensitivity in IDH1 WT cells Murat Gunel Ketu Mishra

23 What about other IDH1/2-mutant tumors? Acute myelogenous leukemias (AML) Similar neomorphic mutations S. Halene Y. Song Image from: Saha et al. (2014) IDH mutations in liver cell plasticity and biliary cancer. Cell Cycle, 13,

24 Validation of mutant IDH1-dependent PARPi sensitivity in vivo Yes, we see this in tumors with endogenous IDH1/2 mutations Chris Corso, MD, PhD Radiation ncology resident Ranjini Sundaram, PhD Yanfeng Liu, PhD

25 Small molecule mutant IDH1 inhibitors reverse BRCAness and eliminate the vulnerability to PARP inhbitors

26 Bench-to-bedside: Testing olaparib in IDH1/2-mutant cancers in the clinic Ranjit Bindra Paul Eder Now enrolling patients with IDH1/2 mutations

27 LAPC Patient - Metastatic Chondrosarcoma (mutant IDH1) Target 2 Baseline Scan Cycle 1 Cycle 2 Images courtesy of Dr. Geoffrey Shapiro, DFCI

28 Conclusions: IDH mutations and cancer ther oncometabolites? ur work provides an explanation: IDH mutants cause genetic instability and promote carcinogenic genomic changes that persist even if you subsequently block the neomorphic enzyme activity KDM4A/B MA? How does inhibiting KDM4A/B impact DNA repair? KDM4A/B demthylate H3K9me3 and H3K36me3 New mechanism for hypoxia-induced New treatment strategy genetic instability? now being tested in clinical trials Sulkowski 2017, Science Translational Medicine

29 Are there other metabolites like 2HG that can inhibit AKG-dependent enzymes? FUMARATE: elevated in hereditary leiomyomatosis and renal cell cancer (HLRCC) due to fumarate hydratase (FH) deficiency SUCCINATE: elevated hereditary paraganglioma and pheochromocytoma (PGL/PC) due to succinate dehydrogenase (SDH) deficiency Sulkowski 2018, Nature Genetics

30 Elevated fumarate and succinate in human cancers lead to increased DNA DSBs by comet assay

31 Deficiency in FH or SDH causes decreased homologous recombination similar to loss of BRCA1 or BRCA2

32 FH or SDH knockdown, or addition of fumarate or succinate to tumor cells, Causes synthetic lethal sensitivity to PARP inhibitors Sulkowski 2018, Nature Genetics

33 FH or SDH knockdown in tumors causes synthetic lethal sensitivity to PARP inhibitors Tumor growth delay assays Sulkowski 2018, Nature Genetics

34 FH deficient HLRCC tumors are sensitive to the PARP inhibitor, laparib in a tumor growth delay assay in mice UK 262 are patientderived, FH-deficient tumor cells Sulkowski 2018, Nature Genetics

35 What is the mechanism for 2HG, fumarate and succinate suppression of HR? Chromatin immune precipitation (ChIP) assays for DNA repair factor recruitment to a site directed DNA DSB at 30 minute intervals. yh2ax U2S-EJDR CTRL 2 yh2ax U2S-EJDR + 30uM DM- Fumarate 2 H3K9me3 MRE11 TIP60 BRCA1 1 0 Z-Score Break ccupany (% Input ChIP) H3K9me3 MRE11 TIP60 BRCA1 1 0 Z-Score Break ccupany (% Input ChIP) prpa (S4/S8) -1 prpa (S4/S8) -1 Rad51 Rad Time post DNA DSB (h) Time post DNA DSB (h) Defective recruitment of DNA repair factors to chromatin at a site of a DNA DS

36 Conclusions: oncometabolites, DNA repair and cancer therapy IDH1/2 mutant tumors: Gliomas, cholangiocarcinomas, chondro sarcomas, melanomas, AML High levels of 2-hydroxyglutarate (2HG) 2HG inhibits KDM4A/B This causes homologous recombination deficiency and sensitivity to PARP inhibitors Clinical trials testing PARP inhibitors for mutant IDH tumors are underway FH deficient hereditary leiomyomatosis and renal cell cancer (HLRCC) tumors have elevated fumarate SDH deficient paraganglioma and pheochromocytoma (PGL/PC) have elevated succinate Fumarate and succinate act like 2HG and suppress HR through KDM4A/B, causing sensitivity to PARP inhibitors Clinical trails are being planned The mechanism is at the level of chromatin and recruitment of DNA repair factors

37 Glazer Laboratory Parker Sulkowski (grad student) Susan Scanlon (Yale MSTP) Sebastian eck (post-doc) Yanfeng Liu (technician) Denise Hegan (lab manager) Raman Bahal (post-doc) Anisha Gupta (post-doc) Audrey Turchick (grad student) Stan yaghire (post-doc) Elias Quijano (Yale MSTP) Adele Ricciardi (Yale MSTP) Yuhong Lu (post-doc) Rachael Putman (undergrad) Alanna Kaplan (Yale MSTP) Funding NIH R35 CA NIH R01 ES Ranjit Bindra Brian Shuch Stephanie Halene Murat Gunel Jing Li