PARP Inhibitors for the treatment of MPNST Sarcoma Research Laboratory University of Texas, MD Anderson Cancer Center PARP Inhibitors for the treatment of MPNST Christine Kivlin, Roman Belousov, Gonzalo Lopez, Quan-Sheng Zhu, Kai-Lieh Huang, Davis Ingram, Keila E. Torres, Alexander J. Lazar, Raphael E. Pollock, Dina Lev Hi I’m christine kivlin I’m a graduate student in dr dina lev’s lab at the sarcoma research center of the university of texas MD anderson cancer center and my project focuses on PARPi for the treatment of MPNST

Malignant Peripheral Nerve Sheath Tumor (MPNST) Accounts for 3–10% of soft tissue sarcomas Up to 1,200 new cases in the U.S. per year High metastatic potential Neurofibromatosis type I (NF1)-related cases (50%) and sporadic (50%) Associated with a precursor lesion (NF1-related, deep neurofibromata) MPNST is one subtype and accounts for 3-10% of STS. Its cell of origin is thought to be the schwann cell. it is a relatively rare but aggressive cancer (up to 1200 new cases per year). Most MPNST will metastasize (mostly to the lung with the liver and brain being less common). 50% of MPNST is sporadic and 50% of cases are NF1 related. MPNST patients are young; the average age at diagnosis is 30

MPNST treatment and outcome Surgical excision is the mainstay of treatment Radiation/chemotherapy =? High rate of local and systemic recurrence -Early detection of MPNST is currently unavailable -and Surgical excision is the mainstay of treatment with the effect of radiation and chemotherapy largely unknown -however, surgical resection is often difficult because of the location of tumors (near large nerves as seen here with an MPNST adherent to the sciatic nerve), there is a high rate of local and systemic recurrence, and patients have usually progressed to metastatic disease by the time of diagnosis This translates to dismal outcomes, for all patients there is less than 40% 5 year survival rate. As seen here. MPNST is highly aggressive, metastasis is fatal. Zou et al., 2009

There is a critical need for novel effective therapies in MPNST -therefore there is a critical need for new targeted therapies in MPNST.

PARP inhibitors for the treatment of cancer PARPi have been used in clinical trials over the past few years for common cancers, role for sarcoma unknown Tumors with DNA repair defects are most highly sensitive -In the past few years much excitement and attention has surrounded PARP inhibitors (which are pharmacological inhibitors of poly ADP ribose polymerase or PARP) for the treatment of cancer --Initial preclinical and clinical work shows tumors with DNA repair defects (especially in HR) as seen in BRCA -/- breast and ovarian cancer, and recently in Ewing’s sarcoma, are highly sensitive to PARPi, through synthetic lethality and possibly in combination with conventional chemotherapy

The goal of our studies was to determine the effects of PARP inhibitors on MPNST in vitro and in vivo The goal of my studies is to evaluate the effect of PARP inhibitors in MPNST in vitro and in vivo

Experimental Models Time (weeks) Tumor volume (mm3) Lopez et al., 2011

PARPi treatment reduces PARP activity MPNST 724 PARP inhibitor (10uM) Relative PARP Activity MPNST 26T PARP inhibitor (10uM) Relative PARP Activity -I first wanted to determine if the level of PARP activity decreased in my cells after the addition of PARP inhibitors using a parp activity assay which is essentially an ELISA that semi-quantitatively detects PAR deposited onto immobilized histone proteins -as you can see here there is a much greater decrease in PARP activity with AZD and ABT vs bsi in both 724 and 26T cell lines -this makes sense because as I just said it Seems BSI201 may not be a true PARP inhibitor, based on its mechanism of action. AZD2281 Olaparib (AstraZeneca) ABT888 Veliparib (Abbott Laboratories) BSI201 Iniparib (Sanofi Aventis)

Decrease in cell proliferation with AZD2281 Control Cell lines 96 hour treatment MDA-231 % cell growth 724 MDA-436 I then looked at the effect of increasing doses of AZD2281 on control cell lines from the literature to ensure this was not a non-specific effect. MCF7 is an ER positive breast adenocarcinoma cell line, as you can see it is more resistant than 724 MDA231 is a BRCA pos breast adenocarcinoma cell line known to be resistant to PARPi. MDA436 is a BRCA neg breast adenocarcinoma cell line known to be sensitive to PARPi TC71 is a ewing’s sarcoma cell line, recently shown in the literature to be sensitive to PARPi. Finally I used Lipo224A which is a liposarcoma cell line used in our lab. I used this cell line to ensure the effect of PARP inhibitors was not due to a phenomenon with our cell lines in our lab. Overall the effect of AZD2281 on 724 more closely resembles the known sensitive cell lines from the literature. AZD2281 (uM)

Decrease in cell proliferation with AZD2281 MPNST Cell Lines 96 hour treatment NSC 724 % cell growth 26T ST88 To establish an effect of AZD2281 on cell proliferation I performed MTS assays with each of my MPNST cell lines using increasing doses of AZD2281 for 96 hours. As you can see MPNST 724 is my most sensitive cell line. Overall there is a dose dependent effect on cell proliferation with AZD treatment and all MPNST cell lines are more sensitive than NSC control. 462 AZD2281 (uM)

AZD2281 treatment decreases colony forming ability MPNST 724 Another aspect of cell growth is clonogenicity. I looked at the colony forming potential of all my MPNST cell lines with AZD treatment, at approx 14 days. Today I am showing representative images from one cell line 724, as well as, a graph of multiple replicate experiments. You can see very few clones at the 2.5uM dose, with no clones at 5 and 10uM. My other MPNST cells have the same trend in response to AZD with less overall sensitivity than 724. control 5.0 10.0 0.6125 1.25 2.5 AZD2281 (uM)

AZD2281 causes G2/M cell cycle arrest MPNST 724 24 hour treatment control 2.5uM AZD 5.0uM AZD 10.0uM AZD G1 G2 G2 G2 G1 G2 G1 S S S G1 S the effect of PARP inhibitors on the Cell cycle is one aspect of cell growth. I see a striking G2/M arrest in 724 with AZD at only 24 hrs after treatment, and a similar but less extensive effect in ST88. G2/M: 22.794% G2/M: 53.217% G2/M: 66.366% G2/M: 79.795%

AZD2281 induces apoptosis MPNST 724 96 hour treatment control 2.5uM AZD 5.0uM AZD 10.0uM AZD I then looked at the effect of AZD2281 on apoptosis. Here i treated 724 with increasing doses of AZD for 96 hours., I see a dose dependent increase in apoptosis in 724 cells. As well as an increase in apoptosis in ST88 with only one dose of AZD used. I see a similar increase in apoptosis with increasing doses of AZD in my other MPNST cell lines. 8% total apoptosis 30% total apoptosis 35% total apoptosis 42% total apoptosis

Effect on tumor growth MPNST Xenograft: Subcutaneous Injection (16 mice) Tumors grow to 5mm Intraperitoneal injection To examine the effect of PARPi on local tumor growth in vivo I utilized our subcutaneous mouse model. To start Mice are injected sub cutaneously with 2 million MPNST 724 cells, we begin treatment once the tumors reached 5mm in size; treatment is intraperitonal AZD2281 injected 50mg/kg for approx.3 weeks at which time the mice are sacrificed and the tumor volume and weight is measured. HPCB=2-hydroxypropyl-cyclodentrin Vehicle (8 mice) PBS+10%DMSO+10%HPCB Treatment (8 mice) 50mg/kg/day AZD2281 Sacrifice mice when vehicle group reaches 1500mm3, measure tumor volume and weight, preserve tissue

AZD2281 abrogates tumor growth MPNST 724 MPNST 724 MPNST 26T * p= 0.0002 p= 0.0002 * * p= 0.0002 The results of the previous schema are shown here. The Treatment group received azd2281 for 24 days at which time the tumor volumes for the control group were approx 1500mm^3 and the mice were sacrificed. Tumors were resected, measured, and weighed. There is a statistically significant decrease in tumor weight and volume with the drug treatment as compared to the vehicle group. P=.000231(for tumor vol) P=.0002 (for tumor weight)

Conclusions MPNST cells are relatively sensitive to PARP inhibition in vitro Decrease in cell proliferation G2/ M cell cycle arrest Enhanced apoptosis Anti-tumor effect of PARPi in vivo Cytostatic effect on tumor proliferation Role of PARPi in combination with chemotherapy preclinical/clinical trials? In summary MPNST cells are relatively sensitive to PARP inhibition Effect of PARPi seen in vitro are: Increase in cell proliferation G2/M Cell cycle arrest And an Increase in apoptosis There is an anti-tumor effect of PARPi seen in vivo this is mostly a Cytostatic effect instead of cytotoxic effect which suggests a potential advantage to identifying a combination treatment for additional anti-MPNST effects

Acknowledgements Finally I would like to acknowledge and thank all my labmates and especially Drs. Torres, Lazar, Pollock and Lev for their help and guidance. Thank you

PARP Function and Inhibition here is a very simplistic view of the classical role of PARP in DNA damage repair specifically BER -Once DNA damage occurs (either nicks, DNA SSBs or DSBs)PARP1 is rapidly recruited and binds to DNA. catalytic activity increases 10-500 fold, resulting in synthesis of PAR chains 15-30 sec after damage. -a large portion of PAR is bound to PARP1 itself which is an automodification rxn, this recruits proteins like: the scaffolding protein (XRCC1) that then assembles and activates DNA base excision repair (other proteins are either directly recruited to PAR, or indirectly recruited because they interact with other PARylated proteins) -in addition, PARylation of PARP1 releases PARP1 from damaged DNA, which provides access for other DNA repair proteins and suppresses further PAR synthesis This activity can be exploited for cancer treatment. -PARP inhibition causes persistent SSBs in DNA that are converted into DSBs. If DNA damage response is intact in cells, both HR and DDR proteins will the repair the DSBs. -However, HR deficient cells or cells with BRCAness are unable to repair the accumulated DSBs caused by PARP inhibition, resulting in collapsed replication forks, chromosome instability and cell death.

PARP Activity Assay AZD2281 ABT888 BSI 201 + - Biotinylated NAD: Histone- coated strip wells AZD2281 ABT888 BSI 201 -Add PARPi of interest -Add cell lysate -Add PARP cocktail, incubate Add TACS Sapphire Add HCl Read Absorbance at 450nm + - NAD -PARP activity assay is essentially an ELISA which semi-quantitatively detects PAR deposited onto immobilized histone proteins in a 96 well format. -Useof histone coated wells is because, PARP catalyzes the NAD-dependent addition of PAR to itself and nuclear proteins such as histones. -assay is ideal for screening of PARP inhibitors 1.Rehydrate the histone-coated wells 2. Add PARP inhibitors of interest to wells (could be in serial dilution to determine IC50)orange is control PARPi supplied from kit (3-aminobenzamide and AG014699 3. Add cell lysate to each experimental well ((controls include a cell lysate alone well; (+) positive control which is a supplied PARP enzyme but no inhibitor(this is an activity control, should represent 100% activity reference point), the negative control without PARP enzyme or PARPi to determine the background absorbance)) 4.Add PARP cocktail into each well, this contains biotinylated NAD which will act as the substrate for the PARP enzyme in the cell lysate and will be incorporated into the product PAR; (incubate, wash to remove excess NAD) 5. Add Strepavidin-HRP, which will bind to biotinylated NAD which has been used for PAR formation, (incubate and wash to remove excess Strep-HRP) 6. Add TACs-Sapphire which is a colorimetric substrate which turns blue in the presence of Horseradish Peroxidase (HRP) 7. Addition of HCl stops the reaction and turns the color yellow, then absorbance is then read at 450nm Biotinylated NAD: Strepavidin HRP:

Complex MPNST Karyotype 724 The chromosome # in this cell line ranged from 47-91 , the modal chromosome # being 58

DNA damage PCR array Results Name Relative Fold Change to NSC Upregulated 724 ST88 462 BRCA1 9.7(± 3.2) 29.9(± 8.7) 18.0(± 6.1) CCNO 10.3(± 4.8) 43.5(± 24.4) 161.6(± 86.7) EXO1 30.7(± 5.2) 32.2(± 5.8) 43.9(± 10.9) FEN1 7.8(± 3.6) 13.1(± 4.3) 12.6(± 3.6) LIG1 19.0(± 6.8) 24.6(± 5.5) 26.6(± 9.9) MSH2 5.4(± 0.6) 5.3(± 0.7) 20.4(± 3.3) NEIL3 10.9(±5.7) 13.7(±10.1) 20.3(±17.5) PARP1 6.6(± 3.6) 12.1(± 6.1) 12.6(± 6.4) POLD3 8.7(± 2.8) 6.8(± 2.7) 6.5(± 2.1) RAD51 11.0(± 7.2) 8.5(± 4.1) 15.6(± 4.1) RAD54L 29.7(± 12.3) 30.4(± 7.6) 38.4(± 11.8) RPA3 5.5(± 0.3) 6.1(± 0.5) 5.5(± 0.1) XRCC2 24.5(± 5.5) 37.8(± 6.41) 30.9(± 3.4) Rad51 37kDa NSC 26T 724 462 ST88 T265 Β-Actin NSC 26T 724 462 T265 ST88 PARP 1 B-Actin