Shifalika Tangutoori

PARP inhibitors: A new era of targeted therapy.

Personalized medicine seeks to utilize targeted therapies with increased selectivity and efficacy in preselected patient cohorts. One such molecularly targeted therapy is enabled by inhibiting the enzyme poly(ADP-ribose) polymerase (PARP) by small molecule inhibitors in tumors which have a defect in the homologous DNA recombination pathway, most characteristically due to BRCA mutations. Olaparib, a highly potent PARP inhibitor, has recently been the approved for ovarian cancer therapy by the FDA and European commission in patients with platinum-sensitive, recurrent, high-grade serous ovarian cancer with BRCA1 or BRCA2 mutations. Currently, clinical trials with several PARP inhibitors are being conducted to assess the toxicities, the efficacies and the benefit of the drugs as monotherapies or combined with radiation or other chemotherapeutic agents, in ovarian, breast, prostate, rectal, lung, pancreatic, peritoneal, head and neck, brain, squamous cell carcinomas and sarcomas, to list a few. In this review, our focus is to outline the emerging molecular mechanisms, preclinical evidence and clinical applications of PARP inhibitors especially in nonBRCA cancers, and review the combination strategies compatible with PARP inhibitor therapy.

Simultaneous delivery of cytotoxic and biologic therapeutics using nanophotoactivatable liposomes enhances treatment efficacy in a mouse model of pancreatic cancer

A lack of intracellular delivery systems has limited the use of biologics such as monoclonal antibodies (mAb) that abrogate molecular signaling pathways activated to promote escape from cancer treatment. We hypothesized that intracellular co-delivery of the photocytotoxic chromophore benzoporphyrin derivative monoacid A (BPD) and the anti-VEGF mAb bevacizumab in a nanophotoactivatable liposome (nanoPAL) might enhance the efficacy of photodynamic therapy (PDT) combined with suppression of VEGF-mediated signaling pathways. As a proof-of-concept we found that nanoPAL-PDT induced enhanced extra- and intracellular bevacizumab delivery and enhanced acute cytotoxicity in vitro. In an in vivo subcutaneous mouse model of pancreatic ductal adenocarcinoma, nanoPAL-PDT achieved significantly enhanced tumor reduction. We attribute this to the optimal incorporation of insoluble BPD into the lipid bilayer, enhancing photocytotoxicity, and the simultaneous spatiotemporal delivery of bevacizumab, ensuring efficient neutralization of the rapid but transient burst of VEGF following PDT. From the Clinical Editor: Most patients with pancreatic ductal adenocarcinoma (PDAC) by the time present the disease it is very advanced, which unavoidably translates to poor survival. For these patients, use of traditional chemotherapy often becomes ineffective due to tumor resistance to drugs. Photodynamic therapy (PDT) can be an effective modality against chemo-resistant cancers. In this article, the authors investigated the co-delivery of a photocytotoxic agent and anti-VEGF mAb using liposomes. This combination was shown to results in enhanced tumor killing. This method should be applicable to other combination of treatments.

Light-Controlled Delivery of Monoclonal Antibodies for Targeted Photoinactivation of Ki-67.

The selective inhibition of intracellular and nuclear molecules such as Ki-67 holds great promise for the treatment of cancer and other diseases. However, the choice of the target protein and the intracellular delivery of the functional agent remain crucial challenges. Main hurdles are (a) an effective delivery into cells, (b) endosomal escape of the delivered agents, and (c) an effective, externally triggered destruction of cells. Here we show a light-controlled two-step approach for selective cellular delivery and cell elimination of proliferating cells. Three different cell-penetrating nano constructs, including liposomes, conjugates with the nuclear localization sequence (NLS), and conjugates with the cell penetrating peptide Pep-1, delivered the light activatable antibody conjugate TuBB-9-FITC, which targets the proliferation associated protein Ki-67. HeLa cells were treated with the photosensitizer benzoporphyrin monoacid derivative (BPD) and the antibody constructs. In the first optically controlled step, activation of BPD at 690 nm triggered a controlled endosomal escape of the TuBB-9-FITC constructs. In more than 75% of Ki-67 positive, irradiated cells TuBB-9-FITC antibodies relocated within 24 h from cytoplasmic organelles to the cell nucleus and bound to Ki-67. After a second light irradiation at 490 nm, which activated FITC, cell viability decreased to approximately 13%. Our study shows an effective targeting strategy, which uses light-controlled endosomal escape and the light inactivation of Ki-67 for cell elimination. The fact that liposomal or peptide-assisted delivery give similar results leads to the additional conclusion that an effective mechanism for endosomal escape leaves greater variability for the choice of the delivery agent.

Nanoformulation of Olaparib amplifies PARP inhibition and sensitizes PTEN/TP53-deficient prostate cancer to radiation

The use of PARP inhibitors in combination with radiotherapy is a promising strategy to locally enhance DNA damage in tumors. Here we show that radiation-resistant cells and tumors derived from a Pten/Trp53-deficient mouse model of advanced prostate cancer are rendered radiation sensitive following treatment with NanoOlaparib, a lipid-based injectable nanoformulation of olaparib. This enhancement in radiosensitivity is accompanied by radiation dose-dependent changes in γ-H2AX expression and is specific to NanoOlaparib alone. In animals, twice-weekly intravenous administration of NanoOlaparib results in significant tumor growth inhibition, whereas previous studies of oral olaparib as monotherapy have shown no therapeutic efficacy. When NanoOlaparib is administered prior to radiation, a single dose of radiation is sufficient to triple the median mouse survival time compared to radiation only controls. Half of mice treated with NanoOlaparib + radiation achieved a complete response over the 13-week study duration. Using ferumoxytol as a surrogate nanoparticle, MRI studies revealed that NanoOlaparib enhances the intratumoral accumulation of systemically administered nanoparticles. NanoOlaparib-treated tumors showed up to 19-fold higher nanoparticle accumulation compared to untreated and radiation-only controls, suggesting that the in vivo efficacy of NanoOlaparib may be potentiated by its ability to enhance its own accumulation. Together, these data suggest that NanoOlaparib may be a promising new strategy for enhancing the radiosensitivity of radiation-resistant tumors lacking BRCA mutations, such as those with PTEN and TP53 deletions. Mol Cancer Ther; 16(7); 1279–89. ©2017 AACR.

Abstract B48: Prostate cancer pre-treatment with nanoformulated Olaparib overcomes radiation resistance

Prostate cancers with PTEN deletions are promising candidates for DNA repair inhibitors such as olaparib and talazoparib. Here we show that radiation-resistant cells and tumors derived from Ptenpc-/-;Trp53pc-/- mice are rendered radiation-sensitive following pre-treatment with liposomal nanoOlaparib. This enhancement in radiosensitivity is accompanied by radiation dose-dependent changes in γ-H2AX expression and is specific to nanoformulated Olaparib alone. In animals, twice-weekly intravenous administration of nanoOlaparib alone results in significant tumor growth inhibition. When nanoOlaparib is administered prior to radiation, we find that a single dose of radiation is sufficient to increase mouse survival time by as much as 10 weeks (study duration = 13 weeks). Using ferumoxytol as a surrogate nanoparticle, magnetic resonance imaging (MRI) studies revealed that nanoOlaparib administration enhances the ability of nanoparticles to accumulate in tumors. Compared to untreated and radiation-only controls, nanoOlaparib-treated tumors showed 18-fold higher nanoparticle accumulation, suggesting that the in vivo efficacy of nanoOlaparib may be potentiated by its ability to enhance its own accumulation in tumors. Citation Format: Anne L. van de Ven, Shifalika Tangutoori, Paige Baldwin, Ju Qiao, Codi Gharagouzloo, Nina Seitzer, John Clohessy, Houari Korideck, G. Mike Makrigiorgos, Robert Cormack, Pier Paolo Pandolfi, Srinivas Sridhar. Prostate cancer pre-treatment with nanoformulated Olaparib overcomes radiation resistance. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B48.

Abstract 4335: Nanoformulations of PARP inhibitors Olaparib and Talazoparib for targeted cancer therapy

Introduction: Poly(ADP-ribose) Polymerase (PARP) plays an important role in a number of DNA repair pathways. PARP inhibitors (PARPi) such as Olaparib and Talazoparib exploit the concept of synthetic lethality by selectively targeting cancer cells with defective DNA repair pathways. These drugs are currently only available in oral form which results in limited bioavailability, poor tumor accumulation, and systemic toxicity. Here we report the development of novel nanoformulations of Olaparib and Talazoparib to allow intravenous or intraperitoneal delivery, providing greater bioavailability and tumor accumulation, while limiting systemic toxicities. Methods: Nanoparticle formulations of Olaparib and Talazoparib were synthesized and tested in vitro and in vivo. Short-and long-term dose response with a panel of ovarian cancer cell lines were conducted. These cell lines include KURAMOCHI, SKOV3, OVSAHO, JHOS2, PA1, COV318, 403 and 404, derived from BRCA2-/-, PTEN-/-, TP53mut mice, and 4306 and 4412, developed from conditional LSL-K-rasG12D/+, PTENloxP/loxP mice. Radiosensitization with NanoOlaparib was tested in the radiation resistant prostate cancer cell line FK01, derived from Ptenpc-/-;Trp53pc-/- mice. In vivo, NanoOlaparib was tested in an IP spread model using 404 cells. Animals were treated IP with NanoOlaparib alone, and in combination with cisplatin. Radiosensitization with NanoOlaparib in vivo was tested in a xenograft model using FK01 cells to mimic castration resistant prostate cancer. Animals were treated biweekly with NanoOlaparib before and after radiation treatment. Results: The murine cell lines 403 and 404 were highly sensitive to this treatment due to the mutations in BRCA2, PTEN, and TP53. 4412 and 4306 showed comparable sensitivity, suggesting that a PTEN deletion confers similar sensitivity to PARP inhibitors as a BRCA2 deletion. PA1 demonstrated high sensitivity to NanoOlaparib which may be attributed to genetic instability. NanoTalazoparib is more potent than NanoOlaparib, resulting in a similar relationship in cell line sensitivity with overall lower IC50’s. Strong synergistic radiosensitization was observed in FK01 cells with NanoOlaparib. Bioluminescence imaging illustrated that NanoOlaparib administered IP daily resulted in a greater inhibition of tumor growth than those treated with oral Olaparib daily. The FK01 xenografts are highly radioresistant with little difference between untreated and radiation only animals. NanoOlaparib delays tumor growth, while the combination of radiation and NanoOlaparib clearly shrinks tumors. Conclusions: Robust nanoparticle formulations of NanoTalazoparib and NanoOlaparib have been successfully developed for in vitro and in vivo studies. These results show that NanoOlaparib and NanoTalazoparib amplify the therapeutic efficacy of PARP inhibition and imply a very promising role for the nanoformulation in ovarian and prostate cancers. Citation Format: Paige Baldwin, Anders Ohman, Jeremy Thong, Shifalika Tangutoori, Anne van de Ven, Rajiv Kumar, Daniela Dinulescu, Srinivas Sridhar. Nanoformulations of PARP inhibitors Olaparib and Talazoparib for targeted cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4335.

In vitro analysis of PARP inhibitor nanoformulations

PARP-l is a DNA repair protein that plays a role in a number of repair pathways and also helps in transcriptional regulation; thus PARP inhibitors (PARPi), such as olaparib and BMN-673, act by inhibiting DNA damage repair. This leads to an accumulation of deleterious mutations leading to genetic instability as a result of a number of cell replications. Currently, olaparib is only available in an oral form and has poor bioavailability, consequently leading to poor accumulation in the tumor due to first-pass metabolism. Therefore, in the present study, an injectable nanoparticle formulation of olaparib was created that offers a delivery route in which the drug would be fully bioavailable in the vasculature, suggesting greater tumor accumulation. Our results illustrated that injectable nanoformulations of olaparib and BMN-673, a next generation PARPi, could be developed, and an efficacy test indicated that BMN-673 is a much more potent PARPi than olaparib. The success of these molecular inhibitors as a monotherapy in inhibiting colony formation suggests enhanced efficacy of these treatments in combination with other therapies, even in tumors which have developed resistance.

Abstract A03: PARP inhibitor nanotherapy for ovarian cancer.

Introduction: Poly-ADP-Ribose Polymerase (PARP) inhibitor therapy exploits a synthetic lethality strategy in ovarian cancers specifically endowed with inherent damage in DNA repair or transcription pathways. Talazoparib and Olaparib are potent PARP inhibitors that are currently indicated for oral inhibitor therapy in several clinical trials for a variety of cancers. Oral administration of these inhibitors typically results in poor bioavailability and tumor accumulation. Here we report the first novel nanoformulations NanoTalazoparib and NanoOlaparib, thus enabling a platform which provides a safe vehicle for parenteral administration specifically targeted to the tumor, thereby increasing the bioavailability while reducing systemic toxicity. Methods: Three nanoparticle (~120nm size) formulations NanoOlaparib, NanoOlaparibPt and NanoTalazoparib, have been successfully formulated and tested in vitro on several cancer cell lines. KURAMOCHI, SKOV3, and OVSAHO were cultured in RPMI + 10% FBS. JHOS2 was cultured in RPMI + 10% FBS +1% Non-Essential Amino Acids. PA1, COV318, 403, 404, 4412, and 4306 were all cultured in DMEM + 10% FBS. 403 and 404 were derived from tumors of BRCA2-/-¬, PTEN-/-, and TP53mut mice. 4306 and 4412 4306 were developed from conditional LSL-K-rasG12D/+/PTENloxP/loxP mice. Dose Response: Cell lines were exposed to either Olaparib or NanoOlaparib concentrations ranging from 0 to 100 µM. Each cell line was treated for a total of four doubling cycles to ensure that the percent viability for each cell line was comparable. Cell viability was ascertained with an MTS assay, to measure the metabolic activity of the cells. Pt synergism: The synergism due to chemosensitization using cisplatin was studied for both therapies using isobolograms developed from a delayed viability assay. Results: In vitro studies PA1 is highly sensitive to NanoOlaparib which may be attributed to genetic instability at 11/13 polymorphic loci, each containing (CA)¬n microsatellites. Microsatellite instability has the potential to cause mutations in critical genes that contain coding repeat sequences. This suggests that the genetic instability in PA1 leads to downstream mutations conferring sensitivity to PARP inhibitors. The murine cell lines, 403, 404 are the next most sensitive group to this treatment due to there triply mutated genomic profile. The high sensitivity of 4412 and 4306 cell lines suggests that PTEN deletion confers similar sensitivity to PARP inhibitors as a BRCA2 deletion. Loss of PTEN has been shown to lead to spontaneous DSBs, chromosomal instability, and defects in homologous recombination. While it was expected that cell lines with BRCA1/2 mutations would be some of the most sensitive to these treatments, the results indicate that BRCA mutations and deletions are just as susceptible as PTEN deletions while high genetic instability shows the greatest sensitivity. NanoTalazoparib is 10-100 times more potent than Olaparib. The cell line dependence is similar to Olaparib except for the overall lower magnitudes. In vivo studies A pilot study was carried out in an endometrial OvCa murine model with KRaS-PTEN deletion to test the nanoformulations for biocompatibility and therapeutic efficacy. Bioluminescence images show tumor suppression of more than a nearly a factor of 3. All formulations were well tolerated. Studies in BRCA2-/-¬/ PTEN-/-/TP53mut GEMM also showed good therapeutic response to i.p. administration. Conclusions: Robust nanoparticle formulations of NanoTalazoparib and NanoOlaparib have been successfully demonstrated for in vitro and in vivo administrations. These results show that NanoOlaparib and NanoTalazoparib amplify the therapeutic efficacy of PARP inhibition and imply a very promising role for the nanoformulation in ovarian and other cancers. This work was supported by the DOD Ovarian Cancer Research Program under Army- W81XWH-14-1-0092. Citation Format: Paige Baldwin, Anders Ohman, Jeremy Thong, Shifalika Tangutoori, Daniela Dinulescu, Srinivas Sridhar. PARP inhibitor nanotherapy for ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A03.

Abstract B35: Nanoformulations of PAPR inhibitors nanoolaparib and nanotalazoparib for targeted cancer therapy

Poly-ADP-Ribose Polymerase (PARP) inhibitor therapy exploits a synthetic lethality strategy in ovarian cancers specifically endowed with inherent damage in DNA repair or transcription pathways. Here we report the first novel nanoformulations of PARP inhibitors NanoTalazoparib and NanoOlaparib, thus enabling a platform which provides a safe vehicle for parenteral administration specifically targeted to the tumor, thereby increasing the bioavailability while reducing systemic toxicity. Methods: Three nanoparticle (∼120nm size) formulations NanoOlaparib, NanoOlaparibPt and NanoTalazoparib, were successfully formulated and tested in vitro and in vivo on several cancer cell lines. Ovarian Cancer cell lines tested include KURAMOCHI, SKOV3, and OVSAHO, JHOS2 PA1, COV318, 403/ 404 (derived from tumors of BRCA2-/-¬, PTEN-/-, / TP53mut mice) and 4306 / 4412 (developed from conditional LSL-K-rasG12D/+/PTENloxP/loxP) mice. IC50s were determined with an MTS assay. Radiosensitization studies with NanoOlaparib were carried out on prostate cancer cell lines LNCAP, PC3, and FK01 (derived from PTEN-/P53- mice tumors). In vivo studies were carried out with iv or ip administration. Results: In vitro studies The murine cell lines, 403, 404 were highly sensitive to this treatment due to the triply mutated genomic profile. The high sensitivity of 4412 and 4306 cell lines suggests that PTEN deletion confers similar sensitivity to PARP inhibitors as a BRCA2 deletion. The PA1 is highly sensitive to NanoOlaparib which may be attributed to genetic instability at 11/13 polymorphic loci. Strong radiosensitization was observed in the prostate cancer cell lines. NanoTalazoparib is 10-100 times more potent than NanoOlaparib. The cell line dependence is similar to NanoOlaparib except for the overall lower magnitudes. In vivo studies In a pilot study in an endometrial OvCa murine model with KRaS-PTEN deletion, bioluminescence images show tumor suppression of more than a nearly a factor of 3. Studies in BRCA2-/-¬/ PTEN-/-/TP53mut OvCa GEMM also showed good therapeutic response to i.p. administration. In vivo studies in prostate cancer models showed greater tumor accumulation and tumor reduction with NanoOlaparib. Conclusions: Robust nanoparticle formulations of NanoTalazoparib and NanoOlaparib have been successfully demonstrated for in vitro and in vivo administrations. All formulations were well tolerated.. These results show that NanoOlaparib and NanoTalazoparib amplify the therapeutic efficacy of PARP inhibition and imply a very promising role for the nanoformulation in ovarian and other cancers. This work was supported by the DOD Ovarian Cancer Research Program under Army- W81XWH-14-1-0092. Citation Format: Paige Baldwin, Ilanchezhian Shanmugam, Shifalika Tangutoori, Anders Ohman, Daniela Dinulescu, Robert Cormack, Mike Makrigiorgos, Srinivas Sridhar. Nanoformulations of PAPR inhibitors nanoolaparib and nanotalazoparib for targeted cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B35.

Abstract AS29: PARP inhibitor nano-therapy in ovarian cancer models

Introduction: Poly-ADP-Ribose Polymerase (PARP) inhibitor therapy exploits a synthetic lethality strategy in cancers specifically endowed with inherent damage in DNA repair or transcription pathways. Olaparib is a potent PARP inhibitors that is currently indicated for oral inhibitor therapy in several clinical trials for a variety of cancers. Oral administration of these inhibitors in general results in poor bioavailability and tumor accumulation. Here we report the first novel nanoformulations customized for olaparib (NanoOlaparib), thus enabling a platform which provides a safe vehicle for intravenous delivery specifically targeted to the tumor, thereby increasing the bioavailability while reducing systemic toxicity. Methods: Two nanoparticle (120nm size) formulations NanoOlaparib and NanoOlaparibPt have been successfully formulated and tested in vitro on several cancer cell lines. Dose response curves over a dynamic range of nanoPARPi therapy on several cell lines PA-1, KURAMOCHI, OVSAHO, SKOV3, and 4306, were generated using MTS assay and EC509s were determined using Prism. The synergism due to chemosensitization using cisplatin was studied for both therapies using isobolograms developed from delayed viability assay. Results: In vitro studies Cell viability studies were carried out with NanoOlaparib and NanoOlaparibPt in OvCa cell lines. The highly Pt-sensitive cell line PA-1 is strongly responsive to NanoOlaparib (blue) and NanoOlaparibPt (grey) although it is not known to carry a germline BrCa mutation. The multi-drug resistant cell line SKOV-3 is also more responsive to NanoOlaparib and combination NanoOlaparibPt. In vivo studies A pilot study was carried out in an endometrial OvCa murine model with KRaS-PTEN deletion to test the nanoformulations for biocompatibility and therapeutic efficacy. Bioluminescence images (Fig. 2) show tumor suppression of more than a nearly a factor of 3. All formulations were well tolerated. Conclusions: A robust nanoparticle formulation of the PARP inhibitor, Olaparib, has been successfully demonstrated. Both chemo sensitization and radio sensitization were studied in PC3, VCaP cell lines. Combinatorial administration of Nano (Olaparib+Cisplatin) showed greater cell death than Cisplatin alone or Cisplatin+ Olaparib/DMSO. We observed a significant enhancement in the cell killing ability (both immediate and delayed) with NanoOlaparib when compared to olaparib alone. Increased tumor accumulation and therapeutic efficacy were observed in prostate and breast cancer GEM models. These results show that NanoOlaparib amplifies the therapeutic efficacy of PARP inhibition and imply a very promising role for the nano-olaparib formulation in ovarian and other cancers. Citation Format: Shifalika Tangutoori, Paige Baldwin, Jamie Medina, Anders Ohman, Daniela Dinulescu, Srinivas Sridhar. PARP inhibitor nano-therapy in ovarian cancer models [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS29.