Sequential therapeutic strategy in targeting ovarian cancerous cells. Tahira Baloch Master Student Dr. Walter Gotlieb’s Lab Lady Davis Institute ,McGill University 8th December 2016

- Pelvic Epithilial Cancer Introduction - Pelvic Epithilial Cancer - Olaparib(FDA approved Drug) - Cisplatin (penicillin of cancer) - Synthetic Lethality (Novel strategy in tumors having deficient pathways) To say small lines with it

What causes Cancer? Cancer arises from the MUTATION of normal genes. A fact which brings about the mutation is called as MUTAGEN. It is thought that several mutations need to occur to give rise to cancer. Mutated genes that cause cancer are called ONCOGENES.

Ovarian Cancer Second most common gynaecological cancer. >75% advanced stage diagnosis. HGSEOC(High Grade Serous Epithilial Ovarian Cancer) is the most common and aggressive form of ovarian cancer.HRD (homologous Recombination Deficiency)is common and thus display high rate of platinum and PARP inhibitory sensitivity. Enlargement of abdomen common sign. Prevention: annual pelvic exam. More patients die of ovarian cancer than from cervical and endometrial combined. Ovarian cancer is the most lethal of all gynecological malignancies, due in part to the diagnosis at an advanced stage caused by the lack of specific signs and symptoms and the absence of reliable tests for screening and early detection

Ovarian Cancer 70% “Complete response” 70-80% Relapse Surgery Chemotherapy 70% “Complete response” 70-80% Relapse Most patients will respond initially to treatment but about 70% of them will suffer a recurrence. Therefore, new therapeutic modalities are urgently needed to overcome chemoresistance observed in ovarian cancer patients Although most patients with EOC experience a reasonable initial clinical response to debulking surgery and chemotherapy, the majority of these patients will not be cured. Approximately 70% will experience a recurrence and this chemoresistance is responsible for the majority of ovarian cancer-related deaths [4]. Presently, there are no available treatments capable of curing recurrent ovarian carcinomas due to their rapid evolution into a chemoresistant disease. It has therefore become essential to introduce new therapeutic modalities that will change response to treatment into cure and salvage these patients.

Trends in Ovarian Cancer Incidence and Mortality Rates (1975-2015) Percentage surviving 5 years 44.6% (2004-2010) When compared to other gynecological cancers, the fatality rate of ovarian cancer surpasses that of cervical and endometrial cancers put together Estimated new cases in 2014 (US) 21,980 % of all new cancer cases 1.3% Estimated deaths in 2014 (US) 14,270 % of all cancer deaths 2.4%

Types of Ovarian Cancer Low grade, indolent Low stage Represents 25% of ovarian cancer Responsible for 10% of ovarian cancer deaths 50% High grade, Aggressive Poor outcome Represents 75% of ovarian cancer Responsible for 90% of ovarian cancer deaths

Risk factors : Advancing age Infertility Endometriosis Family history Inheretid Gene mutation or Genetic susceptibility (8-13% caused by BRCA1 or BRCA2) Ethnicity (>white women,<black women & Asian)

BRCA1 gene BRCA 1 BRCA1 is tumor suppressor gene First cloned in 1994 DNA Repair Genome Stability BRCA1 is tumor suppressor gene First cloned in 1994 Help repair damaged DNA up to 50 % of OC patients have BRCA1gene mutaions. And There is strong link of BRCA1 and genetic stability in ov arian cancer especially in ovarian in breast cancer. As BRCA1 is involved in ds DNA repair , cell cycle arrest, cell death

Types of DNA damage and repair Base excision repair Type of damage: Repair pathway: enzymes: Double- strand breaks (DSBs) Single- breaks (SSBs) PARP Recombinational DNA-PK HR NHEJ BRCA ATM RAD51 Ultraviolet light Ionizing radiation Man-made and natural chemicals Reactive oxygen species most are generated “endogenously” DNA damage can occur in several different forms, including SSBs or double-strand breaks (DSBs).1 In higher eukaryotes, genomic stability is essential for healthy functioning and survival. DNA damage may induce mutations and can lead to cell death via apoptosis.2 Therefore, several repair mechanisms have evolved to maintain the integrity of the genome.1 Base excision repair (BER) is a key pathway in the repair of SSBs and is reliant on the enzyme poly(ADP-ribose) polymerase (PARP).1 For DSB repair, there are two predominant pathways: Homologous recombination (HR) that involves a protein kinase, ataxia-telangiectasia mutated (ATM) Non-homologous end-joining (NHEJ) that requires DNA-dependent protein kinase (DNA-PK).2 HR is the most accurate mechanism for repairing DSBs, whereas NHEJ is rarely error-free.2 Abbreviations on slide: AGT, O(6)-alkylguanine-DNA alkyltransferase; ATM, ataxia telangiectasia mutated; MLH1, MutL homolog; MSH2, MutS homolog; XP, xeroderma pigmentosum References 1. Jackson SP, Bishop CL. Drug Discovery World 2003; Fall: 41-45. 2. Jackson SP. Biochem Soc Trans 2001; 29: 655-661. References 1. Jackson SP, Bishop CL. Drug Discovery World 2003; Fall: 41-45. 2. Jackson SP. Biochem Soc Trans 2001; 29: 655-661.

PARP Poly(ADP-ribose) polymerases (PARPs) are enzymes known as key regulators of cell survival and cell death Single-strand DNA break repair through base excision repair pathway is mediated by PARP Poly(ADP-ribose) polymerases (PARPs) are enzymes that have been recently recognized as key regulators of cell survival and cell death. Drugs that inhibit PARP-1 help fight cancers caused by mutations in BRCA1 and BRCA2. In one study, the PARP inhibitor olaparib was also able to shrink tumors in ovarian cancer patients who did not have BRCA mutations. Clinical trials of this type of drug are being done to see who will benefit most from them.

Trapping PARP-DNA complexes. PARP INHIBITORS : Act by 2 mechanisms, Catalytic inhibitors. Trapping PARP-DNA complexes. PARP inhibitors were taught to work primarily by blocking PARP enzyme activity,thus preventing the repair of DNA damage and ultimately causing cell death. But now scientists established that PARP inhibitors have an additional mode of action;localizing PARP proteins at sites of DNA damage. The trapped PARP protein, DNA complexes are highly toxic to cells because they block DNA replication. Trapped PARP-DNA complexes are more toxic to cell than the unrepaired single strand DNA break that accumulates in the absence of PARP activity,indicating the PARP inhibitors act as PARP poisons. Two classes of PARP inhibtors: Catalytic inhibitors that act mainly to inhibit PARP enzyme activity and do not trap PARP proteins on DNA. Dual inhibitors that both block PARP enzyme activity and act as PARP poison

PARP inhibitor: Olaparib (AZD 2281)

Timeline for PARP inhibitors development:

CISPLATIN: (penicillin Of Cancer) Also known as CIS-diamminedicholorplatinium (CDDP) 1st member of a class of platinium-containing anti-cancer drug. Administered as IV as short-term infusion for solid malignancies. Effective against various types of cancers including sarcomas,lymphomas and carcinomas(ovarian cancer)

MECHANISM OF ACTION: Cisplatin bind to DNA and cause a critrical structure change in the DNA- a bend of 45 degree. 3 main component in mechanism of action: Cisplatin DNA HMG protein With cell membrane cisplatin goes inside thru active transport and some with passive diffusion,once in nucleus cisplatin form an abduct with its two consecutive gaunine with startds of DNA.the molecule loses its CL atom and form bond with nitrogen atom of target gaunine,PT binds more effectively with N as compare to Cl. It is this abduct induce Dna bends that allow the binding of protiens which coontains HMG domain.once protein is bound to DNA insert the wedgeb like fennel group .phe37 is bind in wedge like reated by nend.The tightly bend HMG causes destacking of neocleotides bases resulted in DNA helix becoming cinks.in dis way,CS followed as monkey renche as DNA repair system.because of this HMG protein DNA is unable repair.and causes apoptosis in cancerous cells.

Synthetic Lethality Most pathways involved in breast and ovarian cancer are related to GENOMIC INSTABILITY. BRCAm cancer may be vulnerable to PARPi,as part of synthetic lethality strategy. CITY OF hope is currently investigating ABT-888 (parpi) in combination with cisplatin in BRCAm ,The results are promising.

Synthetic lethality Non fuctional BRCA + PARP inhibition BER HR BER HR Inhibition of PARP-1 activity prevents the recruitment of DNA repair enzymes and leads to failure of SSB repair and accumulation of SSBs. During the S-phase of the cell cycle, the replication fork is arrested at the site of an SSB, which then degenerates into a DSB. In normal cells, this triggers activation of the HR pathway to repair the DSB.1 Abbreviation on slide: PNK 1, polynucleotide kinase 1 Reference 1. Helleday T et al. Cell Cycle 2005; 4: 1176-1178. Synthetic lethality

Future Prospects: We hope that the licensing of olaparib( for BRCAm) is the first step in it making a real difference to the lives of patients with ovarian cancer worldwide. 

My Goal: Tumors having epigenetically silenced or mutated BRCA1 can be effectively treated with sequential administration of PARP inhibitors(olaparib) Causing Synthetic Lethality And Then followed by : cytotoxic agent(cisplatin) Causing Remaining Cancerous cells to die.

Thank you for the attention!