John R. Macdougall

Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926).

Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the A-ring system generated three series of analogues with improved potency and/or solubility. Lead compounds from each series were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result, complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after daily oral administration of 40 mg/kg of compound 28.

Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.

An infrared dye-conjugated virus-like particle for the treatment of primary uveal melanoma

The work outlined herein describes AU-011, a novel recombinant papillomavirus-like particle (VLP) drug conjugate and its initial evaluation as a potential treatment for primary uveal melanoma. The VLP is conjugated with a phthalocyanine photosensitizer, IRDye 700DX, that exerts its cytotoxic effect through photoactivation with a near-infrared laser. We assessed the anticancer properties of AU-011 in vitro utilizing a panel of human cancer cell lines and in vivo using murine subcutaneous and rabbit orthotopic xenograft models of uveal melanoma. The specificity of VLP binding (tumor targeting), mediated through cell surface heparan sulfate proteoglycans (HSPG), was assessed using HSPG-deficient cells and by inclusion of heparin in in vitro studies. Our results provide evidence of potent and selective anticancer activity, both in vitro and in vivo. AU-011 activity was blocked by inhibiting its association with HSPG using heparin and using cells lacking surface HSPG, indicating that the tumor tropism of the VLP was not affected by dye conjugation and cell association is critical for AU-011–mediated cytotoxicity. Using the uveal melanoma xenograft models, we observed tumor uptake following intravenous (murine) and intravitreal (rabbit) administration and, after photoactivation, potent dose-dependent tumor responses. Furthermore, in the rabbit orthotopic model, which closely models uveal melanoma as it presents in the clinic, tumor treatment spared the retina and adjacent ocular structures. Our results support further clinical development of this novel therapeutic modality that might transform visual outcomes and provide a targeted therapy for the early-stage treatment of patients with this rare and life-threatening disease. Mol Cancer Ther; 17(2); 565–74. ©2017 AACR.

Abstract 1770: Viral-based nanoparticles: a new therapeutic approach for anticancer therapy

One of the hurdles to effective cancer treatment is tumor specific targeting of drugs. We have observed, both in vitro and in vivo, that Human papillomavirus-derived nanoparticles (HPV-NPs) have an innate tropism for tumors. HPV9s primary attachment factors in infection are heparan-sulfated proteoglycans (HSPG) and as tumors often upregulate HSPGs at the extracellular matrix, this may be a contributing factor for HPV9s tumor tropic nature. Here we present data to support the use of HPV-NPs in the treatment of ovarian carcinoma and uveal melanoma with α-particle emitting radiation (227Th) and photodynamic therapy (PDT), respectively. We first explored in vivo tumor targeting using HPV-NPs delivering a plasmid expressing the luciferase gene. Luciferase signal was detected in SKOV-3 tumor bearing, but not control animals, and this signal was only present in tumor tissue as revealed by ex vivo imaging. This finding prompted us to explore the therapeutic effect of delivering 227Th. The agent AU-010 was constructed by conjugation of a chelator to the HPV-NPs. Th-227 was then used to radiolabel the conjugated HPV-NPs. Animals were injected with SKOV-3 tumor cells, and 10 days following implant, treatment was initiated with three weekly administrations of either AU-010 (100 nCi), free chelated-227Th (100 nCi), unlabeled HPV-NPs or saline. HPV-NP targeting of 227Th to tumors provided a significant survival advantage over the control arms, demonstrating the therapeutic potential of HPV-NPs. Currently, follow-up experiments are evaluating optimized chelators in additional animal models. We next explored the use of HPV-NPs in photodynamic therapy with the development of AU-011. AU-011 is an HPV-NP conjugated to ≈500 molecules of infrared dye, IR700 (LiCor), and when irradiated with 690 nm near-infrared light generates reactive oxygen species that are specifically and immediately lethal to dye bound cells. This technology was initially validated using Trastuzumab as a targeting agent (Mitsunaga et al, Nat Med 17(12):1685-91, 2011). We compared the cell binding activity of AU-011 and Trastuzumab-IR700 on SKOV-3 ovarian cancer cells in vitro and observed a higher binding capacity of AU-011. We next compared the killing activity of the two agents. Trastuzumab-IR700 was found to have an IC50 value of 1.6 nM when activated with 32 J of 690 nm light. By comparison, AU-011 was found to have an IC50 value of 5.6 pM when activated with 16 J of 690 nm light. Thus, AU-011 appeared to be considerably more potent than Trastuzumab-IR700. Finally, we extended these observations to a panel of uveal melanoma cell lines. We chose uveal melanoma because PDT with 690 nm light is FDA approved and used regularly in the ophthalmic clinic. When applied on the melanoma cell lines, AU-011 was found to show high cell binding and potent cell killing. Currently, proof-of-concept in vivo studies of AU-011 in uveal melanoma are underway, paving the way to clinical development for this indication. Citation Format: Rhonda Kines, Debaditya Bhattacharya, John R. MacDougall, Diane Milenic, Martin W. Brechbiel, Elisabet de los Pinos, John T. Schiller. Viral-based nanoparticles: a new therapeutic approach for anticancer therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1770. doi:10.1158/1538-7445.AM2014-1770

Abstract 3285: Hedgehog pathway inhibition delays regrowth of ovarian cancer following paclitaxel and carboplatinum only if initiated immediately after completion of chemotherapy

The high mortality associated with ovarian cancer is attributed to the lack of any reliable early detection method, unknown pathogenesis of the disease, and the development of recurrent and chemoresistant tumors. Current efforts have focused on the identification of therapeutics that may be used independently or in combination with current chemotherapeutic regimens to reduce tumor volume. To date, limited research has focused on preventing or delaying disease recurrence. The Hedgehog (Hh) signal transduction pathway is inactive in most adult cells. Malignant activation of the Hh pathway through the signaling protein Smoothened (Smo) occurs in a broad range of cancers, including ovarian. IPI-926 is a potent orally delivered small molecule that targets the Hh pathway by inhibiting Smo. Recent studies from our laboratory provide evidence that IPI-926 slows serous ovarian cancer growth in a primary human tumor xenograft model. More importantly, IPI-926 delays the resurgence of tumor growth typically observed after cytoreduction with paclitaxel and carboplatinum (T/C) treatment. Our current objective was to assess whether this effect of IPI-926 required that the Smo inhibitor be administered during a critical window following T/C treatment to prevent the resurgence of tumor growth. To test our hypothesis, mice bearing human ovarian cancer xenografts were treated with vehicle or T/C. T/C treatment was withdrawn following significant reduction (30-50%) in tumor volume. The original vehicle treated cohort was divided into 2 arms which then either received IPI-926 or continued on vehicle for the duration of the experiment. Mice in the T/C cohort were divided into 3 groups. Group 1 received vehicle alone (T/C-Vehicle), group 2 received IPI-926 immediately following the last T/C dose (T/C-IPI-926) and group 3 received no vehicle or IPI treatment for 14 days following the last T/C dose (Window). Group 3 was then maintained on IPI-926 treatment starting at day 15 post-T/C withdrawal. The withdrawal of T/C led to a dramatic increase in tumor volume in the T/C-Vehicle group. As previously observed, tumor growth inhibition was maintained in mice receiving IPI-926 immediately following the cessation of T/C treatment. In contrast, delaying the administration of IPI-926 following T/C prevented the suppression of tumor growth as evidenced by the increased tumor volume. Our data suggest that blocking Hh pathway activity immediately following chemotherapy maintains and prolongs the inhibitory effect of chemotherapy on ovarian tumor growth. The absence of an IPI-926-mediated inhibition of tumor resurgence following a 14-day delay in treatment supports the concept that there is a critical period for tumor re-establishment. Our data, along with those of others, suggest that the early stages of ovarian tumor re-growth may be dependent on Hh pathway signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3285. doi:1538-7445.AM2012-3285

Abstract 979: The anti-tumor activity of the Smoothened inhibitor IPI-926 in models of residual disease is time dependent

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Although response rates can be high for some solid tumors and hematologic malignancies following treatment, cure rates are low and there is a need to extend the duration of response and overall survival after remission. This relapse is attributed to the presence of residual malignant cells that remain undetected after primary treatment and in many instances the presence of these residual tumor cells eventually leads to disease recurrence and shortened survival. The role of the Hedgehog (Hh) pathway in cancer has been well established, with both ligand-independent signaling due to genetic mutations, as well as ligand-dependent signaling, in which Hh signaling occurs either directly to the tumor cells or to the tumor microenvironment being observed. Moreover, it has been shown that Hh ligand produced by tumor cells acts on stromal cells and inhibition of this interaction results in alterations to the tumor-associated stroma and significant inhibition of tumor growth. IPI-926 is a potent and selective Hh pathway antagonist that inhibits the key signaling membrane protein Smoothened (Smo). IPI-926 delays tumor regrowth post-cytoreduction with either standard of care chemotherapy or targeted therapy in pre-clinical xenograft models of small cell lung (SCLC), non-small cell lung (NSCLC) and prostate cancer. Data reported here show that the activity of IPI-926 in this post-therapy setting exhibits a strict time-dependence, or “efficacy window”. In both the Lx-22 SCLC and H1650 NSCLC models, delaying the time between cytoreductive therapy and initiation of IPI-926 treatment resulted in significant loss of anti-tumor activity. To investigate the molecular consequences underlying this loss of activity, RT-PCR analysis of tumors from different time points post therapy was performed. These results showed upregulation of human Hh ligand in the tumors in response to cytoreductive therapy with subsequent increased Hh signaling in the murine-derived stromal cells. IPI-926 abrogated this signaling. In addition, histological and immunohistochemical analysis demonstrated that IPI-926 induced significant changes in the tumor microenvironment post therapy. Taken together, these data suggest that the administration of IPI-926 post cytoreductive therapy could potentially be beneficial in the residual disease setting in multiple cancer types and warrants further investigation. Moreover, the timing of IPI-926 administration post therapy is critical for this benefit. IPI-926 is currently being evaluated in three Phase 2 clinical trials designed to explore multiple approaches to target ligand-dependent activation of the Hh pathway: 1) a randomized Phase 2 trial in combination with gemcitabine in patients with metastatic pancreatic cancer; 2) a randomized Phase 2 in patients with metastatic or locally advanced chondrosarcoma; and 3) a single-arm Phase 2 trial in patients with myelofibrosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 979. doi:1538-7445.AM2012-979

Abstract B145: The novel Hedgehog pathway inhibitor IPI-926 delays metastatic spread and provides a survival benefit in an experimental model of pancreatic liver metastasis.

Metastasis, or the spread of disseminated tumor cells to and colonization of distal sites, is a major cause of cancer mortality. The Hedgehog (Hh) signaling pathway is normally involved in embryogenesis; however recent evidence suggests a role for this pathway in cancer and metastasis. Thus, inhibitors of Hh signaling may have potential utility as novel targeted anti-tumor therapies. IPI-926 is a novel, selective, small molecule that antagonizes the Hh pathway by binding to the Smoothened receptor, the major upstream facilitator of Hh signal transduction, and is currently in phase 2 clinical trials for pancreatic cancer, chondrosarcoma and primary myelofibrosis. Administration of IPI-926 in combination with gemcitabine inhibits incidence of metastases in a KRAS/p53 transgenic model of pancreatic cancer (Olive et al., 2009), supporting an integral role for the Hh pathway in this process. To further explore the activity of IPI-926 in the setting of metastasis, we developed an experimental model of pancreatic cancer liver metastasis where luciferase-tagged tumor cells were implanted in the liver via an intra-splenic injection, followed by splenectomy, resulting in liver metastases. Tumor burden, as assessed by bioluminescence and survival were employed to evaluate IPI-926 activity utilizing different dosing regimens. While treatment of tumor-bearing animals with established disease provided no survival benefit, IPI-926 administered on the day of cell implant provided modest but reproducible increase in survival. However, prophylactic administration of IPI-926 beginning 14 days (d) prior to cell implant resulted in a significant decrease in disease burden and enhanced survival. The activity was dependent on the schedule of IPI-926 administration. Treatment initiated 14d prior to cell implant was more effective than 7d before cell implant, and treatment started only 2d prior to cell implantation showed no difference compared to treatment initiated on the day of cell implant. Histological analysis of pre-treated vs control livers confirmed decreased metastatic nodule formation. In addition, analysis of serum harvested from IPI-926 14d pre-treated vs control animals showed down-regulation of components of the VEGF, PDGF and MMP pathways, suggesting that IPI-926 modulation of these pathways may be involved in the observed effects on metastatic spread. The activity of IPI-926 was also evaluated in the setting of sunitinib enhancement of metastases (Ebos et al., 2010) in the above model. As has been shown previously, short, high-dose sunitinib administration resulted in striking increases in disease burden and shortening of survival compared to control animals. However, when IPI-926 was administered 14d prior to cell implant and sunitinib treatment, disease burden and survival were returned to the levels observed in the control group. In summary, IPI-926 administration delays metastasis in an experimental model of pancreatic liver metastasis and in response to high-dose anti-angiogenic therapy. These results provide rationale for the evaluation of IPI-926 as adjuvant/neoadjuvant therapy to help control metastatic spread post surgical resection in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B145.

Abstract 1601: Hsp90 inhibition results in a significant delay in tumor progression in a model of emerging EGFR TKI resistance in non-small cell lung cancer

Heat shock protein 90 (Hsp90) plays a role in regulating the stability of key cancer-causing proteins through its role as a protein chaperone. Proteins chaperoned by Hsp90, known as client proteins, include cancer-causing forms of ALK, BCR-ABL, EGFR, FLT3 and HER2. Infinity is developing two drug candidates in its Hsp90 chaperone inhibitor program: IPI-504 (retaspimycin hydrochloride), an intravenously-administered small molecule, and IPI-493, which is administered orally. EGFR tyrosine kinase inhibitors (TKIs) are an effective treatment for lung cancer patients with activating mutations in EGFR. After a dramatic initial response, however, most patients become resistant to drug treatment and progress. In about half of these cases, resistance is due to a second point mutation in EGFR (T790M). It is believed that in at least some of these cases, the TKI resistance mutations are pre-existing and that treatment with TKIs selects for the resistant cells. In an effort to model the emergence of resistance to TKIs from pre-existing mutations, we developed a novel in vivo model, where gefitinib treatment initially leads to tumor regression followed by rebound of tumor growth and outgrowth of drug resistant clones containing the T790M mutation. We show that in this model, treatment with IPI-493 alone and IPI-493 following gefitinib resulted in tumor growth inhibition of 61 and 77%, respectively, when compared with gefitinib treatment alone. Treatment with IPI-493 alone also resulted in a significant delay in time to tumor progression with ∼40% of animals still on study 45 days following tumor implant; all animals treated with either vehicle or gefitinib had been removed due to tumor progression. Interestingly, treatment with IPI-493 following gefitinib resulted in an even more impressive delay in time to progression, with >50% of animals still on study on day 65 post-implant. These results suggest that further studies with Hsp90 inhibitors in EGFR mutant NSCLC patients who have been pre-treated with a TKI may be warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1601. doi:10.1158/1538-7445.AM2011-1601

Abstract 5045: The Hh inhibitor IPI-926 inhibits relapse of a non-small cell lung cancer xenograft model following treatment with an EGF-R targeted tyrosine kinase inhibitor

The Hedgehog (Hh) pathway plays a key role in multiple types of cancer, either by mutational activation of the pathway in tumor cells or by ligand-dependent signaling to the tumor cells or paracrine signaling between tumor cells and the tumor microenvironment. We have previously shown in a primary model of SCLC that the Hh pathway inhibitor IPI-926 prevents tumor relapse after regression of the tumor with chemotherapy (Travaglione et al., AACR 2009). To investigate whether tumor regression with a targeted agent could elicit a similar response as that seen with chemotherapy, two NSCLC lines (H1650 and HCC827), with activating mutations in the EGFR were evaluated. NSCLC cell lines bearing mutant EGFR are exquisitely sensitive to EGFR kinase inhibitors. Upon treatment with gefitinib in vitro, HCC827 increased the expression of Indian and Sonic hedgehog ligands 10-fold, as measured by qRT-PCR. When grown in mice as subcutaneous xenografts, H1650 tumors regressed following treatment with the EGFR inhibitor gefitinib (40 mg/kg every day) for 7 days. The gefitinib-treated mice were then administered either vehicle or IPI-926 at 40 mg/kg every other day for three weeks, and in this setting IPI-926 treatment led to a 65% decrease (p=0.0104) in tumor volume compared to vehicle-treated mice. Conversely, tumors that had not been regressed with the EGFR TKI showed no response to the Hh pathway inhibitor, similar to our previous results in SCLC with chemotherapy. Studies are ongoing to define the mechanism of action of IPI-926 in this setting. When grown in mice as subcutaneous xenografts, these tumor cells produce Shh ligand in conjunction with stromal Gli-1 expression in the murine stroma. Stromal Gli-1 expression is inhibited by treatment with IPI-926, indicating that Hh ligand produced by NSCLC tumor cells signals to stroma in a paracrine manner. Together with our previous results in SCLC, these data suggest that therapeutic inhibition of the Hh signaling pathway might be an important strategy to extend progression free survival in patients who initially respond to therapy but later relapse, and that this paradigm is applicable to multiple cancer types and treatment modalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5045.

Abstract LB-374: The Hh inhibitor IPI-926 enhances tumor perfusion and nab-paclitaxel activity in a pancreatic xenograft model

Malignant activation of the Hedgehog (Hh) pathway is associated with multiple tumor types. In certain cancers, such as pancreatic, a paracrine role for the Hh ligand has been described, wherein cancer cells produce Hh ligand that activates the Hh pathway in the surrounding stroma. Consistent with this model, IPI-926, a potent and selective Smoothened (Smo) inhibitor, blocks Hh signaling in the mouse stroma -but not in the cancer cells- of several pancreatic xenograft models. We recently published (Olive, Science 2009) that IPI-926 increases vascular perfusion and enhances gemcitabine drug delivery to tumors in a genetically engineered mouse model of pancreatic cancer (KPC) leading to an increase in overall survival. To determine if similar effects could be observed in xenograft models of human pancreatic cancer, experiments were designed to study the combination of IPI-926 with nab-paclitaxel, an agent that has recently demonstrated anti-tumor activity in pancreatic cancer (Van Hoff, ASCO 2009). While IPI-926 had no single agent activity in the L3.6pl human pancreatic xenograft model, it enhanced the activity of nab-paclitaxel from 61% tumor growth inhibition (nab-paclitaxel alone) to 83% tumor growth inhibition (nab-paclitaxel plus IPI-926, p=0.0048). Tumor IHC analysis of phosphohistone 3 showed a higher frequency of cells arrested at the late G2/M phase in the IPI-926 plus nab-paclitaxel group versus nab-paclitaxel alone (p=0.02). One possible explanation for the synergistic effect of a combination of IPI-926 and nab-paclitaxel is that IPI-926 affects the mouse stroma and increases tumor perfusion and nab-paclitaxel accessibility to the tumor. Tumor perfusion was directly measured in IPI-926 treated and untreated animals using contrast enhanced ultrasound. In tumor bearing animals treated with IPI-926 for 7 days, the ultrasound data showed greater tumor perfusion with IPI-926. On average, the peak time for contrast agent levels decreased from 11.0 seconds to 4.75 seconds in the vehicle versus IPI-926 treated animals, respectively, (p=0.0321). These data suggest that the mechanism of synergy between IPI-926 and nab-paclitaxel is likely enhanced drug delivery to the tumor through the effect of IPI-926 on the stroma. Studies are ongoing to measure nab-paclitaxel and paclitaxel levels in IPI-926 treated and untreated tumors, and to investigate these findings with the KPC in situ mouse model of pancreatic cancer. These preclinical data provide a strong rationale for evaluating the Hh inhibitor IPI-926 not only with the current standard of care, gemcitabine, but with emerging new potential therapies like nab-paclitaxel in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-374.