DNA damage

Question 1

introduction

Answer 1

DNA stores genetic information of cell
DNA damage threatens integrity and stability of genome
occurs frequently = 50,000 to 70,000 DNA damage events/cell/day
induced by a variety of exogenous and endogenous sources
cells have evolved sophisticated mechanisms to repair DNA damage = different pathways to repair different types of damage
failure to repair = mutations, genome instability, cancer

Question 2

types, causes, pathways

Answer 2

see table

Question 3

sensing ssbreaks + study

Answer 3

• Single-stranded breaks (SSBs) detected by poly (ADP-ribose) polymerase 1 (PARP1)

• Catalyses transfer of ADP-ribose from NAD to target proteins on chromatin (e.g. histones, PARP-1 itself) at site of DNA breaks
• Results in formation of long branched chains of poly (ADP-ribose) = promote recruitment of proteins crucial for SSBR (e.g. XRCC1, DNA ligase III)

• Trucco et al (1998) = PARP KO sensitises cells to mutagens

• Exposed WT and PARP-/- mouse embryonic fibroblasts to sublethal dose of alkylating mutagen methylmethanesulfonate (MMS)
• PARP-/- cells displayed significant defects in SSB repair = reduced ability to proliferate (as measured by methyl-3H-thymidine incorporation), accumulation in G2/M phases of cell cycle (as assessed by flow cytometry), increase in micronuclei (marker of chromosome breaks)
• Transient expression of PARP, through transfection with plasmid encoding human PARP, restored DNA repair ability

Question 4

sensing dsbreaks + condition

Answer 4

• Double-stranded breaks are detected by ATM kinase

• Recruited and activated at DSBs
• Phosphorylates various downstream proteins (e.g. p53, CHK2, BRCA1) = halts cell cycle progression, promotes recruitment of repair enzymes to damage

• ATM is mutated in the condition ataxia telangiectasia (A-T), associated with:

• Increased sensitivity to agents that induce DSBs (e.g. ionising radiation)
• Little/no hypersensitivity to other forms of DNA damage
• Presents with ataxia, progressive neurodegeneration

Question 5

MMR

Answer 5

Many mismatches are corrected by exonuclease component of DNA polymerase = proofreading ability
If not  mismatch repair steps in to facilitate correction of base-base mismatches

1. MutSα (MSH2-MSH6) or MutSβ (MSH2-MSH3) recognises and binds to mismatches
2. Other molecules are recruited to the DNA = e.g. MutLα (PMS2-MLH1), MutH, proliferating cell nuclear antigen (PCNA), replication factor C (RFC)
3. MutH cleaves backbone in vicinity of mismatch  slides along DNA in direction of mismatch, liberating strand to be excised
4. Exonuclease 1 (EXO1) digests ssDNA segment containing erroneous base
5. DNA polymerase repairs the single-stranded gap
6. DNA ligase seals new strand in place

Question 6

mutsa and mutsB redundancy

Answer 6

• Two complexes are partially redundant
• MutSα is much more abundant than MutSβ = repairs most mismatches, MutSβ only required for repair of larger insertion-deletion loops (IDLs)
• Explains different tumour phenotypes in KO mice
- MSH2 KO mice (de Wind, 1995) = most severe phenotype  most develop lymphomas at a very early age
- MSH6 KO mice (de Wind, 1999) = less severe
- MSH3 KO mice (Edelmann, 2000) = not tumour-prone

Question 7

MMR in cancer

Answer 7

Lynch syndrome
• autosomal dominant condition associated with germline mutation in MLH1, MSH2, MSH6, PMS2
• characterised by defective mismatch repair, elevated rate of single nucleotide changes, microsatellite instability, hypermutation
• patients at high risk of a range of cancer types = particularly colorectal, endometrial, gastric and ovarian
• cancers typically have high TBM = respond well to immunotherapy

DNA mismatch repair pathway genes also frequently mutated in somatic cancers
Chalmers et al (2017)
• performed comprehensive genomic profiling of 100,000+ patient tumours = huge sample!
• identified novel mutation hotspot in PMS2 promoter  mutated in 10% of melanoma cases
• associated with significantly increased tumour mutational burden = good predictor of immunotherapy response
• failed to definitively show if mutations are causal = further experiments required to elucidate effect of promoter mutations

Question 8

NHEJ vs HR

Answer 8

see table

Question 9

NHEJ steps

Answer 9

NON-HOMOLOGOUS END JOINING
• Key steps = synapsis (juxtaposition of 2 DNA ends), end processing and ligation
1. SYNAPSIS
• Ku70-Ku80 heterodimer binds to DNA ends = holds ends in close proximity, prevents end resection
• DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruited = phosphorylates proteins at DNA break, forms scaffold to attract other NHEJ components
- E.g. XRCC4, DNA ligase IV, XLF

2. END PROCESSING
   • ‘Dirty’ ends are processed by Artermis, nucleases and other end processing factors = trim single-stranded overhangs to produce blunt ends that can be ligated together
   • Polymerases (e.g. DNA polymerase lambda) fill the gaps
3. LIGATION
   • DNA ends are ligated by the XRCC4-DNA ligase 4 complex

Question 10

VDJ recombination

Answer 10

NHEJ can be useful! = important for VDJ recombination
• Random rearrangement of variable (V), joining (J) and diversity gene segments in B and T lymphocytes  generates variants of heavy and light chains of Ig and TCRs
• Requires deliberate formation of DSB and repair by error-prone NHEJ
• Drastically increases diversity of antibody and TCR repertoires

Question 11

defects in NHEJ

Answer 11

Defects in NHEJ often result in
• Radiosensitivity
• Severe combined immunodeficiency, resulting from impaired VDJ recombination
• Little hypersensitivity to agents that do not induce DSBs
e.g. radiosensitive severe combined immunodeficiency (mutations in Artemis)
ligase IV syndrome (developmental and growth defects, lymphomas)

Question 12

HR steps

Answer 12

Exchange of homologous segments between 2 DNA molecules

1. DSB detected by ATM kinase
2. Gap widened by exonucleases (e.g. MRN complex) = facilitates sister chromatid strand invasion, recruitment of repair factors
3. Generates 3’ end single-stranded DNA overhangs
4. RAD51 recognises and binds to 3’ single-strand ends = binding influenced by other proteins such as replication protein A (RPA)
5. DNA/RAD51 filament invades homologous DNA
6. RAD51 catalyses strand-exchange events = forms D-loop structure which then forms a double Holliday junction (after RAD52 captures second end by DNA annealing)
7. Holliday junction resolved and cleaved by helicases and nucleases

Question 13

cohesins in HR

Answer 13

Cohesins have been implicated in the repair of double-stranded breaks (DSBs) by homologous recombination (HR). This was illustrated in a study by
Bauerschmidt et al (2010)10
- siRNA-mediated knockdown of SMC1 increases the radiosensitivity of HeLa cells (as measured by colony formation)
- only moderate = but perhaps due to inefficiency of siRNA knockdown or fact that some cells were in G1
- repair of yH2AX DSB foci induced by X-rays during late S phase and G2 (high CENP-F staining and interphase-like DAPI staining) was significantly slower in cells depleted of SMC1 than controls

It is thought that cohesins encourage homologous recombination by establishing sister chromatid cohesion at the site of the DSB, helping to keep the DSB in close proximity to its undamaged sister chromatid to encourage strand invasion and promote sister chromatid HR.

Question 14

global cohesin loading study

Answer 14

Evidence that cohesin loading in response to DNA damage is not site specific, but genome-wide
Unal et al (2007)
- Induced double-stranded break on chromosome 3 in budding yeast cells, by introducing site-specific HO endonuclease into genome under inducible promoter, in addition to 2 HO cleavage sites on chromosome 3
- Cohesion reporter (tandem array of Lac operators that be visualised by LacI-GFP) indicated that DSB on chromosome 3 prevented loss of S-phase cohesion on chromosome 1 and 3  indicative that cohesion occurs even on unbroken chromosomes in response to DSB

Question 15

Rad21 + DNA damage

Answer 15

· The cohesin subunit Rad21 (also known as Scc1) plays a critical role in repair of DNA double-strand breaks
o This was first discovered by Phipps et al. (1985) when they reported that mutated Rad21 rendered S. pombe cells hypersensitive to UV light or infrared radiation.
· siRNA experiments have shown its important, because Rad21 dysfunction appears to play a role in cancer:
· Expression studies by Atienza et al (2005) revealed a 1.25-2.5-fold increased expression of RAD21 gene in human breast cancer cell lines compared to normal breast tissue.
o Cells transfected with RAD21-specific siRNA had reduced Rad21 levels and reduced proliferation compared to controls.
o Moreover, breast cancer cell sensitivity to two DNA-damaging chemotherapeutic agents was increased after inhibiting RAD21 expression; and cells transfected with siRNA against RAD21 showed ~60% survival compared with control cells.
· Hence RAD21 could be a novel target for developing cancer therapeutics that can potentially enhance the anti-tumour activity of chemotherapeutic agents via inducing DNA damage.

Question 16

SMC5-SMC6 and DNA damage

Answer 16

Whilst SMC5-SMC6 complexes remain much less understood than cohesins and condensins, evidence indicates that they play important roles in responses to DNA damage. For example, yeast possessing mutations in any of the subunits of the SMC5-SMC6 complex often display hypersensitivity to agents that can induce DNA damage, such as radiation, UV light and the alkylating agent MMS. Like cohesins, SMC5-SMC6 complexes are recognised to play vital roles in sister chromatid HR following DSBs. This was illustrated in a study by Di Piccoli et al (2006)11, who induced double stranded breaks into specific sites in the genome of yeast cells. Chromatin immunoprecipitation revealed that SMC6 was rapidly recruited to sites localised 4-5kB either side of DSBs, with SMC6 binding to these sites increasing by as much as 5-7-fold. They also found that the repair of DSBs by sister chromatid recombination was reduced by 4-fold in cells possessing SMC6 mutations, whilst the rate of spontaneous gross chromosomal rearrangements was increased by 100-fold.

This study indicates that the SMC5-SMC6 complex plays an important role in facilitating error-free recombination between sister chromatids; however, it fails to reveal the mechanisms by which this is achieved, which remain poorly understood. The SMC5-SMC6 complex is thought to aid repair by establishing sister chromatid cohesion (independent of cohesin), and by guiding the free ends of DSBs to appropriate regions of the nucleus, helping to shield the ends from other regions of homology in the genome to encourage faithful sister chromatid recombination. There is also some evidence to suggest that SMC5-SMC6 recruits cohesin to the DNA in response to damage. This was explored by Potts et al (2006)12, who found that the knockdown of the SMC5-SMC6 subunit MMS21 blocked the recruitment of cohesin to double-stranded breaks, whilst depletion of SCC1 did not have the same effect on SMC5-SMC6 recruitment. (CHIP)
Later evidence contradicts this (Wu et al, 2012)
- Revealed that siRNAs used to deplete mms21 triggered premature sister chromatid separation
- Using different siRNAs to deplete Smc5 and Smc6 that did not have this effect did not prevent cohesin recruitment to damage sites

Question 17

choice between NHEJ and HR

Answer 17

determined by competition between 53BP1 (inhibits end resection = NHEJ) and BRCA1 (promotes end resection = HR)

in G1, 53BP1 phosphorylated by ATM = recruits PTIP and RIP1 to DNA = suppress end-resection by BRCA1 = NHEJ

in S/G2, BRCA1 prevents phosphorylation of 53BP1 = prevents recruitment of RIP1 = end resection promoted = HR

BRCA1 also interacts with BRCA2 and RAD51 to promote strand invasion and recombinase activity

Question 18

evidence that BRCA1 antagonises 53BP1

Answer 18

Bunting (2010)
found that 53BP1 KO in homozygous BRCA1 mutant mice attenuates phenotype of mutation
significantly reduced onset of mammary tumorigenesis
also facilitated 10-fold reduction in chromosomal aberrations and improved survival (measured using FC) of BRCA-deficient B cells stimulated to divide ex vivo for 3 days
also relieved sensitivity to PARP inhibition

loss of 53BP1 also increased HR
treated WT, BRCA1 mutant and BRCA1 mutant 53BP1 KO cells with IR and stained with Rad51 antibody = marker of HR
only 6.8% of BRCA1 deficient cells showed foci vs 30% of mutant KO

Question 19

DNA repair in oncology

Answer 19

DNA repair in oncology
• Cancer cells often sacrifice specific DNA damage response pathways to accelerate accumulation of genetic changes
• E.g. BRCA1/2 genes are mutated in many cases of hereditary breast and ovarian cancer  cells become HR-deficient
• Baert et al (2020)
- Reduced BRCA1 expression in human breast epithelial cell line using silencing RNA
- Led to a 58% reduction in RAD51 foci, indicative of reduced HR, as well as an increase in micronuclei
• Makes cells particularly susceptible to drugs that inhibit alternative DNA repair pathway  studying protein components involved in DNA repair has proven valuable strategy in searching for novel therapeutic targets

Question 20

PARP inhibitors

Answer 20

• PARP inhibitors can be used to treat BRCA-deficient cancers
• Without PARP, single-stranded breaks are not detected  SSBs persist in DNA  may degenerate into double-stranded breaks when DNA is replicated  DSBs cannot be accurately repaired by HR so instead repaired by error-prone NHEJ  frequent chromatid breaks  cell death
• Limited effects on normal cells = slower rate of replication, HR still intact so cells can repair SSBs efficiently
• Known as synthetic lethality = inactivation of both pathways (SSBR and HR) is lethal, deficiency in just 1 is not

• PAPRi also effective against PTEN-deficient tumours (e.g. prostate cancers), which exhibit downregulation of HR component Rad51

Question 21

evidence that BRCA-deficient cells are sensitive to PARPi

Answer 21

Farmer et al (2005) = BRCA-deficient cells are sensitive to PARP inhibitors
• Reduced PARP activity in WT and BRCA1/2 deficient embryonic stem cells using small-molecule inhibitors
• Effects on BRCA-deficient cells = rapid and irreversible
- Induced profound arrest of cells in G2/M phases
- Induced apoptosis  FACS analysis showed that many cells stained positive for annexin V, which binds phosphatidylserine = marker of apoptosis when expressed in outer leaflet of plasma membrane
- Reduced cell survival in clonogenic assays
- Induced frequent chromatid breaks and aberrations
• BRCA-deficient cells much more sensitive to effects than WT cells = 57-fold and 133-fold enhanced sensitivity in BRCA1 and BRCA2 deficient cells respectively
• Inhibitors induced formation of RAD51 foci in dose-dependent manner in WT cells, no foci seen in BRCA-deficient cells regardless of dose = PARPi induce damage normally repaired by mechanism involving RAD51 and requiring BRCA1/2  HR
• Also effective in vivo
- Transplanted WT or BRCA2-deficient embryonic stem cells into athymic mice = formed teratocarcinomas
- PARPi robustly inhibited tumour formation in mice transplanted with BRCA2-deficient cells, but not in mice injected with WT

Question 22

efficacy in breast cancer

Answer 22

• Efficacy in breast cancer = Chang et al (2021) metanalysis

• 6 RCTs comparing PARPi to conventional chemotherapy
• PARPi associated with significantly improved PFS and ORR, but significantly increased risk of grade 3/4 thrombocytopenia
• Limited number of studies

Question 23

resistance to PARPi

Answer 23

Resistance to PARPi = more than 40% of BRCA1/2 deficient patients fail to respond to PARPi
• PARPi become ineffective if tumour cells can restore HR
• Most common mechanism is reactivation of BRCA1 and BRCA2 = secondary frameshifts can realign amino acid sequence and restore function
• Other mechanisms
- Loss of 53BP1 and related proteins
- Loss of Shieldin factors
- Loss of CTC/Pol alpha
- Loss of DYNLL1/ATMIN
- Drug efflux from cells

Question 24

evidence for reactivation of BRCA1/2

Answer 24

Edwards et al (2008)
• Derived PARPi-resistant cells from human pancreatic cancer cell line CAPAN1, which carries BRCA2 mutation, resulting in production of a truncated protein
- Cells could competently perform HR, unlike non-resistant CAPAN1 cells  acquired ability to form RAD51 nuclear foci after DNA damage (e.g. by X-rays)
• Looked at whether resistant cells expressed WT BRCA2
- Performed WB using antibodies recognising N-terminal (present in mutant BRCA2) or C-terminal (absent from mutant BRCA2)  resistant clones expressed BRCA2 species of similar size to WT BRCA2, containing both N and C-terminal epitopes
- Comparative genomic hybridisation in combination with FISH found that resistant cells carried BRCA2 alleles with deletions resulting in elimination of original mutation and restoration of ORF
• Reconstitution of BRCA2-deficient cells with revertant BRCA2 alleles from resistant cells rescued PARPi sensitivity and HR deficiency in vitro
• Identified similar mutations (deletion of original + restoration of IRF) in tumours from 2 patients with ovarian cancer resistant to carboplatin treatment

Question 25

NER steps

Answer 25

1. DNA damage binding proteins detect nucleotide changes and form complex around damage site
2. Transcription factor IIH (XPB and XPD), XPG, and XPF-ERCC1 dimer recruited
3. TFIIH unwinds double helix, XPG and XPF-ERCC1 use exonuclease activity to create incisions to remove DNA damage = encompass area of ~30 nucleotides
4. TFIIH released from DNA
5. Replication protein A and CPA bind = verify DNA damage and keep damaged DNA in place until repair commences
6. DNA polymerase recruited by replication factor C and PCNA = synthesizes complementary DNA to undamaged strand
7. DNA ligase completes repair

Question 26

GGR vs TCR

Answer 26

Two types of NER = equally efficicent
Global genome repair (GGR)
- can occur anywhere in the genome
- helix-distorting damage is recognised by XPC and partners RAD23B and CETN2
Transcription-coupled repair (TCR)
- Facilitates preferential repair of transcribing strand
- Stalling of RNA polymerase at transcriptionally active genes  recruitment of Cockayne syndrome proteins A and B (CSA and CSB)
- More constitutive than GGR

Question 27

Xeroderma pigmentosa

Answer 27

Defects in XP proteins cause the genetic disease xeroderma pigmentosum
• Caused by mutations in XP proteins involved in NER = unable to repair DNA lesions induced by UV irradiation
• Rare disease = 1 in 100,000
• Patients present with severe photosensitivity, skin cancers (average age of onset = 8 years), dry skin, pigmentation treatments, neurological abnormalities
• No treatment = avoidance of sunlight is best preventative measure
• Patients usually die in their 30s
• One mutation that causes XP is XPF

Tian et al (2004) = some XPF function is essential for life
• Used homologous recombination gene-targeting to induce LOF mutation in XPF gene into murine ESCs  used to generate chimeric mice  heterozygous bred to produce homozygotes
- Mutation entirely abolished XPF expression in homozygous cells (RT-PCR)
- XPF KO mice exhibit extreme growth retardation, die soon after birth, enlarged nuclei in liver cells
- Same phenotype as ERCC1 KO mice = confirms interaction
• Also found that fibroblasts isolated from XPF-deficient mouse embryos showed hypersensitivity to UV light and alkylating agent = consistent with role of XPF in NER

Question 28

Cockagne syndrome

Answer 28

Cockayne syndrome
• Associated with defects in transcription-coupled NER due to mutations in CSA or CSB  impede recovery from blocked transcription  increased cell death after DNA damage
• S+S = early cessation of growth and development, severe progressive neurodecline, frailty average lifespan of 12 years
• Cancer is very rare in CS patients  cells with low levels of DNA damage that block transcription and are not dealt with by other repair systems are eliminated before they can give rise to tumours

Question 29

BER

Answer 29

Damaged base is removed whilst SPB is kept intact

Causes of base damage

1. Reactive oxygen species (e.g. hydroxyl radicals)
2. Deamination (e.g. adenine  hypoxanthine, mutagenic as hypoxanthine pairs with cytosine rather than thymine)
3. Alkylation
4. Hydrolysis
5. Damaged DNA recognised by one of 11 DNA glycosylases, which cleave glycosidic bond to excise damaged base from sugar backbone = produces apurinic (AP) site
6. Repaired through short or long patch BER

Question 30

short-patch repair vs long-patch repair

Answer 30

Short patch

1. AP-endonuclease cleaves phosphodiester chain 5’ to AP site
2. AP-lyase activity of DNA polymerase B removes deoxyribose phosphate
3. DNA polymerase B then inserts correct nucleotide based on complementarity
4. DNA ligase III (LIG3), and XRCC1 connects deoxyribose of replacement nucleotide to backbone

Long patch

1. AP-endonuclease cleaves phosphodiester chain 5’ to AP site
2. PCNA acts as scaffold
3. Pol/ perform displacing synthesis = generate oligonucleotide flap by extension
4. Flap endonuclease 1 (FEN1) removes 5’ flap sequence
5. Oligonucleotide ligated to DNA by DNA ligase I (LIG1)

See diagram

Question 31

BER glycosylases study

Answer 31

Klungland et al (1999) = are glycosylases really necessary?
Created homozygous OGG1 KO mice = lack specific DNA glycosylase required to excise most frequent mutagenic base lesion (8-oxoG) induced by ROS
- mice were viable but accumulate abnormal levels of 8-oxoG in genomes
- only moderately elevated spontaneous mutation rate in nonproliferative liver (measured by HPLC-ECD), no increase in tumour incidence, no marked pathology
- extracts of OGG1 KO mouse tissues cannot excise damaged base in vitro = indicates that a different glycosylase is not compensating for loss of OGG1
- But significant slow repair of 8-oxoG lesions seen in OGG1 KO cell line
- Suggests that other mechanisms exist to rectify DNA damage and maintain low endogenous mutation frequency in absence of DNA glycosylases = e.g. transcription-coupled repair

Question 32

ssbreak repair

Answer 32

1. PARP recognises DNA break and assembles poly(ADP-ribose) chains
2. Recruits components of SSB repair complex
   - XRCC1
   - DNA ligase I and III
   - polymerase β
   - DNA end processing enzymes (including polynucleotide kinase phosphatase (PNKP), aprataxin)
3. SSB carries out three major functions
   - Repairs DNA ends, restoring 3’-OH and 5’phosphate structures of SPB that are often damaged or removed during SSBs, due to their reactive nature = diverse range of enzymes required due to variety of possible damage motifs
   - Damaged or missing nucleotides replaced by DNA polymerases (predominately pol β) through short patch repair (inserting single nucleotides) or long patch repair (replacing small sections of nucleotides overlying the damage)
   - Ligation of sugar phosphate backbone by DNA ligases

Question 33

XRCC1 in ssbreak repair

Answer 33

XRCC1 = molecular scaffold, interacts with both DNA and enzymes to regulate and speed up repair process
Thompson et al (1982) = revealed importance of XCCR1 activity in SSB
- XRCC1-deficient hamster cell lines = show reduced rate of SSB repair and increased susceptibility to mutagens, compared to WT cells

Question 34

aprataxin in ssbreak repair

Answer 34

Constitutive part of DNA repair ligase complexes, removes DNA adenylate adducts
Acts as proof-reader of abortive DNA ligation
Hirano et al (2007) = evaluated effects of aprataxin in XPA-UVDE cells, which repair UV-induced damage exclusively by long-patch SSBR
- Used siRNA to knockdown APTX  immunoblot analysis revealed siRNAs significantly suppressed APTX expression  resulted in significantly delayed repair of cyclobutene pyrimidine dimers (CPD) (measured by fluorescence microscopy)
- Transient expression of siRNA-resistant APTX restored impaired repair

Defective in ataxia with oculomotor apraxia 1 (AOA1) = cerebellar ataxia, oculomotor apraxia, confined to wheelchair by early adulthood
Effects are confined to neurons
- low regenerative capacity
- high levels of oxidative stress so frequent DNA damage
- high transcriptional demand
- no replication-coupled HR repair in postmitotic neurons

Question 35

using aprataxin to predict clinical response to TOPO1 inhibitors

Answer 35

Dopeso et al (2010) = tumour aprataxin levels predict clinical response of colorectal cancer to treatment with topoisomerase I inhibitor irinotecan
Irinotecan forms stabilises complex between TOP1 and DNA  DNA strand breaks  replication arrest + apoptosis
Only 20-30% of patients show objective response to irinotecan = urgent need to develop mechanisms to discriminate patients likely to respond
Study + findings
• first used microarray analysis and qRT-PCR to measure expression of >9000 genes in 30 colorectal cancer cell lines
- excellent correlation between aprataxin mRNA levels + growth inhibition and apoptosis induced by irinotecan analog
• then used IHC to assess levels of aprataxin in duplicate tumour samples from 135 patients with metastatic colorectal cancer treated with irinotecan-based chemo
- low aprataxin levels correlated with longer progression-free and overall survival (36 vs 19 months), remaining significant after multivariate adjustment
• Suggests potential as biomarker  needs to be independently confirmed in larger data set
• Why? More aprataxin  more efficient repair of SSB induced by irinotecan  poor response