Targeting the ATR Kinase in Cancer Therapy 2017 Chabner Colloquium - - PowerPoint PPT Presentation

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Targeting the ATR Kinase in Cancer Therapy 2017 Chabner Colloquium - - PowerPoint PPT Presentation

Nov 11, 2023 226 likes •1.04k views

Targeting the ATR Kinase in Cancer Therapy 2017 Chabner Colloquium October 30, 2017 Lee Zou MGH Cancer Center Harvard Medical School Disclosure Consultant/advisory role: Loxo Oncology DNA Damage and Replication Stress Response Pathways

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Targeting the ATR Kinase in Cancer Therapy

Lee Zou MGH Cancer Center Harvard Medical School

2017 Chabner Colloquium October 30, 2017

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Disclosure

  • Consultant/advisory role: Loxo Oncology
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DNA Damage and Replication Stress Response Pathways

Double Strand Breaks Stressed fork Sensors Transducers Effectors Cell cycle arrest Apoptosis Senescence (DSBs) Telomere maintenance Chromatin structure Transcription DNA repair DNA replication

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The checkpoint pathways

DSBs, replication stress

Yeast Human

DSBs DSBs, replication stress PIKKs Mec1-Ddc2, Tel1 ATM CHKs Chk1, Rad53 Chk2 ATR-ATRIP Chk1 Mediators Mrc1 Tof1 Csm3 Brca1 Claspin Timeless Tipin Rad9 Effectors Pds1, Cdc20... p53, Brca1, Nbs1, FANCD2, Cdc25s, RPA...

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A model of ATR activation in response to DNA damage and replication stress

TopBP1 TopBP1 Resection TopBP1 TopBP1 9-1-1 complex 9-1-1 complex Rad17 complex

Adapted from Zou & Elledge 2003 Science

ETAA1 ETAA1

Chromatin Chromatin

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Is the ATR checkpoint a good target for cancer therapy?

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Normal cells Tumors Genomic instability ATR Checkpoint

ATR is required for cancer cells to survive genomic instability

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Normal cells Genomic instability Tumors ATR Checkpoint

ATR is required for cancer cells to survive genomic instability

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Is the ATR checkpoint a good target for cancer therapy? Inhibition of the ATR checkpoint may be beneficial to therapy in specific contexts.

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ATR inhibition could be therapeutically beneficial in specific contexts

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What other cancer-specific vulnerabilities can be targeted by ATR inhibition?

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PARP inhibitors selectively kill BRCA1/2-deficient cells

Nature 2005 Nature 2005

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How do PARP inhibitors selectively kill BRCA1/2-deficient cells?

Lord and Ashworth Nature Med. 2013

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FAD-approved PARP inhibitors are used for treatments of BRCA-deficient ovarian cancer

Olaparib (AstraZeneca) Approved in 2015 for advanced ovarian cancer with BRCA mutations Approved in 2017 for maintenance therapy of ovarian cancer Rucaparib (Clovis) Approved in 2016 for advanced ovarian cancer with BRCA mutations Niraparib (Tesaro) Approved in 2017 for maintenance therapy of ovarian cancer with or without BRCA mutations

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Resistance to PARPi is a clinical challenge

Dalton et al. 2015 The American Journal

  • f Hematology/Oncology
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Can we overcome the PARPi resistance in BRCA-deficient cells?

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Functions of BRCA1/2 in the DNA damage response

Homologous recombination (HR)

Rad51

Resection

  • Mre11
  • Exo1?
  • Dna2?
  • BLM/WRN?

BRCA1 BRCA2

Schlacher et al. 2011 Cell Protection of stalled replication forks

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Many ways to acquire PARPi resistance

  • Restoration of BRCA1/2 reading frames
  • Loss of PARP1
  • Up regulation of efflux pump

Restoration of HR

  • Loss of 53BP1, RIF1, REV7, Artemis (increased resection)
  • Loss of KU (NHEJ)

Restoration of fork protection

  • Loss of PTIP, MLL3/4, CHD4
  • Loss of PARP1
  • Overexpression of RADX

Loss of drug or drug targets

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Do PARPi-resistant BRCA-deficient cancer cells have a common vulnerability that can be targeted?

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Development of BRCA1-deficient cell lines that are resistant to PARPi

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PARPi resistance is not caused by loss of PARP1

  • r up regulation of efflux pump
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PARPi resistance is not caused by restoration of BRCA1

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Multiple proteins implicated in PARPi resistsance are altered in PARPi-resistant cell lines

Restore fork protection? Restore HR?

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The ATR checkpoint pathway is transcriptionally up regulated in PARPi-resistant lines

G2/M Checkpoint ATR Checkpoint DNA repair & Checkpoint DNA repair & Checkpoint

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ATRi preferentially kills PARPi-resistant BRCA1- deficient cells

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ATRi and PARPi are more synergistic in PARPi-resistant BRCA1-deficient cells than in BRCA1-proficient cells

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ATRi broadly overcomes PARPi resistance in BRCA1-deficient cancer cell lines

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ATRi prevents the emergence of PARPi resistance in BRCA1-deficient cancer cells

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How does ATRi overcome the PARPi resistance in BRCA1-deficient cancer cells?

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Rad51 focus formation is partially restored in some but not all PARPi-resistant BRCA1-deficient cells

The activity to form Rad51 foci is either maintained or partially restored in PARPi-resistant cells Homologous recombination (HR)

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PARPi-resistant cells partially bypass BRCA2 but not PALB2 and BRCA2 for Rad51 focus formation

BRCA1-deficient cancer cells partially bypass BRCA1 but not PALB2 PARPi-resistant, BRCA1-deficient cancer cells remain dependent

  • n PALB2 and BRCA2
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PARPi-resistant cells rely on PAL2 and BRCA2 for survival in PARPi

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ATRi blocks Rad51 focus formation when BRCA1 is bypassed by 53BP1 loss

ATR is required for HR even when BRCA1 is bypassed

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ATRi blocks Rad51 focus formation PARPi-resistant BRCA1-deficient cancer cells

ATR is required for the residual HR in PARPi-resistant, BRCA1-deficient cancer cells

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ATRi blocks BRCA2 localization to DSBs in PARPi-resistant BRCA1-deficient cancer cells

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ATR is required for BRCA1-independent recruitment of PALB2 and BRCA2

Partial bypass

  • f BRCA1

PALB2 and BRCA2 remain indispensible ATR is required for PALB2-BRCA2 recruitment

BRCA1-deficient cancer cells (PARPi sensitive or resistant) The residual HR activity is ATR-dependent and required for the resistant cells to survive in PARPi

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Rad51 focus formation is partially restored in some but not all PARPi-resistant BRCA1-deficient cells

Restoration of HR is not an obligated requirement for PARPi resistance?

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Is the function of BRCA1 in fork protection restored in PARPi-resistant cell lines? The residual HR activity in PARPi-resistant cells is necessary but not sufficient for resistance What else is driving PARPi resistance?

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DNA fiber assay to monitor degradation of stalled replication forks

Sequential labeling of newly synthesized DNA HU Stalling of replication forks Stalled forks are protected Stalled forks are unprotected (BRCA1/2-deficient cells) Nucleolytic degradation

  • f nascent DNA

CIdU IdU Schlacher et al. 2011 Cell

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PARPi-resistant cells regain the protection of stalled forks in the absence of BRCA1

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ATRi reactivates Mre11-mediated fork degradation in PARPi-resistant cells

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Stable association of Rad51 with stalled forks is required for protection against Mre11

Rad51

Resection

  • Mre11
  • Exo1?
  • Dna2?
  • BLM/WRN?

BRCA1

?

ATR?

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ATRi blocks the stable association of Rad51 with chromatin and stalled forks

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ATRi blocks the stable association of Rad51 with chromatin and stalled forks

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ATRi blocks the stable association of Rad51 with chromatin and stalled forks

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ATRi reactivates fork degradation in PARPi-resistant BRCA2-deficient cancer cells

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ATRi overcomes PARPi resistance by blocking BRCA1- independent Rad51 loading at DSBs and stalled forks

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Why are PARPi-resistant BRCA-deficient cells more sensitive to ATRi than BRCA-proficient cells?

Efficiency of Rad51 loading/stabilization

  • +

+ +

low ATRi PARPi sensitivity high low BRCA-proficient BRCA-deficient, PARPi-sensitive (e.g. UWB1) BRCA-deficient, acquired PARPi resistance A threshold for significant PARPi sensitivity

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What other cancer-specific vulnerabilities can be targeted by ATR inhibition?

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Cells under high replication stress are sensitive to ATR inhibition

Cell 2013 Mol Cell 2015

Replication stress ATR ssDNA Replication catastrophe

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What causes replication stress in cancer cells?

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APOBEC Family of Cytosine Deaminases

Holmes et al. 2007 TIBS Swanton et al. 2015 Cancer Discovery

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APOBEC-Signature Mutations Are Prevalent in a Subset of Cancers

Breast, Bladder, Head & Neck, Lung Cancers…

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APOBEC3A/B are Mutation Drivers in Multiple Cancer Types

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APOBEC3A/B May Act During DNA Replication

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APOBEC3A/B May Act During DNA Replication

Haradhvala et al. Cell 2016

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Do APOBEC3A/B induce DNA replication stress?

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Inducible Expression of APOBEC3A Activates ATR But Not ATM

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The Activation of ATR by APOBEC3A Is Dependent on UNG2

C U Abasic site (AP site) APOBEC3A UNG2

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APOBEC3A Expressing Cells are Sensitive to ATRi

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APOBEC3A Expressing Cells are Uniquely Sensitive to ATRi

ATRi HU MMC

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APOBEC3A Expressing Cells are Uniquely Sensitive to ATRi

ATRi ATMi DNA-PKi

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Why is APOBEC-induced replication stress unique? Why are APOBEC-expressing cells sensitive to ATRi?

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ATRi induces DSBs during DNA replication in APOBEC3A expressing cells

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ATRi induces replication catastrophe in APOBEC3A expressing cells

ssDNA Replication catastrophe

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APOBEC3A AP sites ATR ssDNA

ATR Counteracts APOBEC-induced Replication Stress

Replication catastrophe

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APOBEC3A AP sites ATR ssDNA Replication catastrophe

ATR Counteracts APOBEC-induced Replication Stress

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ATR Counteracts APOBEC-induced Replication Stress ??

What is driving ssDNA accumulation and replication catastrophe after ATRi treatment?

APOBEC3A AP sites ssDNA Replication catastrophe

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UNG2 is required for the ATRi-induced replication catastrophe in APOBEC3A expressing cells

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ATR inhibition leads to accumulation of AP sites at replication forks in A3A expressing cells

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AP sites may impede DNA polymerases and lead to ssDNA accumulation

AP site ssDNA Zhao et al. 2004 NAR

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A ssDNA and APOBEC driven feed-forward loop that generates AP sites and ssDNA

ssDNA APOBEC AP sites Polymerase stalling ATR

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A ssDNA and APOBEC driven feed-forward loop that generates AP sites and ssDNA

ssDNA APOBEC AP sites Polymerase stalling Replication catastrophe

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Is the endogenous APOBEC activity in cancer cells sufficient to induce replication stress?

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An in vitro assay to measure APOBEC3A/B activity in cancer cells

Burns et al. 2013 Nature

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APOBEC3A/B activity varies in different cancer cell lines

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APOBEC3A/B-dependent basal Chk1 phosphorylation in cancer cells

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Endogenous APOBEC activity in cancer cells is sufficient to render cells susceptible to ATRi

Hi-APOBEC Low-APOBEC

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The unique replication stress imposed by APOBECs renders cancer cells susceptible to ATR inhibition

Hi-APOBEC Low-APOBEC ssDNA APOBEC AP sites Polymerase stalling ATR

ssDNA APOBEC AP sites Polymerase stalling

ATR

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The unique replication stress imposed by APOBECs renders cancer cells susceptible to ATR inhibition

Hi-APOBEC Low-APOBEC

ssDNA APOBEC AP sites Polymerase stalling

ssDNA APOBEC AP sites Polymerase stalling Replication catastrophe

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Thanks to…

Lee Zou lab Remi Buisson Stephanie Yazinski Lilian Kabeche Jian Ouyang Dominick Matos David Moquin Hai Dang Nguyen Tribhuwan Yadav Jiamin Zhang Marie-Michelle Genois

Mike Birer (MGH) Shyamala Maheswaran (MGH) Cyril Benes lab (MGH) Daniel Haber (MGH) Sridhar Ramaswamy (MGH) Mike Lawrence (MGH/Broad) Andre Nussenzweig (NIH) Junjie Chen (MDACC)