Background Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer death in the USA by 2030. Immune checkpoint inhibitors fail to control most PDAC tumors because of PDAC’s extensive immunosuppressive microenvironment and poor immune infiltration, a phenotype also seen in other non-inflamed (ie, ‘cold’) tumors. Identifying novel ways to enhance immunotherapy efficacy in PDAC is critical. Dipeptidyl peptidase (DPP) inhibition can enhance immunotherapy efficacy in other cancer types; however, the impact of DPP inhibition on PDAC tumors remains unexplored. Methods We examined the effects of an oral small molecule DPP inhibitor (BXCL701) on PDAC tumor growth using mT3-2D and Pan02 subcutaneous syngeneic murine models in C57BL/6 mice. We explored the effects of DPP inhibition on the tumor immune landscape using RNAseq, immunohistochemistry, cytokine evaluation and flow cytometry. We then tested if BXCL701 enhanced anti-programmed cell death protein 1 (anti-PD1) efficacy and performed immune cell depletion and rechallenged studies to explore the relevance of cytotoxic immune cells to combination treatment efficacy. Results In both murine models of PDAC, DPP inhibition enhanced NK and T cell immune infiltration and reduced tumor growth. DPP inhibition also enhanced the efficacy of anti-PD1. The efficacy of dual anti-PD1 and BXCL701 therapy was dependent on both CD8+ T cells and NK cells. Mice treated with this combination therapy developed antitumor immune memory that cleared some tumors after re-exposure. Lastly, we used The Cancer Genome Atlas (TCGA) to demonstrate that increased NK cell content, but not T cell content, in human PDAC tumors is correlated with longer overall survival. We propose that broad DPP inhibition enhances antitumor immune response via two mechanisms: (1) DPP4 inhibition increases tumor content of CXCL9/10, which recruits CXCR3+ NK and T cells, and (2) DPP8/9 inhibition activates the inflammasome, resulting in proinflammatory cytokine release and Th1 response, further enhancing the CXCL9/10-CXCR3 axis. Conclusions These findings show that DPP inhibition with BXCL701 represents a pharmacologic strategy to increase the tumor microenvironment immune cell content to improve anti-PD1 efficacy in PDAC, suggesting BXCL701 can enhance immunotherapy efficacy in ‘cold’ tumor types. These findings also highlight the potential importance of NK cells along with T cells in regulating PDAC tumor growth.

Abstract PDAC is typically resistant to chemotherapy and immunotherapy; therefore, novel strategies are needed to enhance therapeutic response. BXCL701 is a well-studied inhibitor of dipeptidyl peptidases 4, 8, 9, and Fibroblast Activation Protein, and has been postulated to work through cytokine induction and macrophage pyroptosis. We examined the effects of BXCL701 and/or PD1 therapy in murine models of PDAC. In the mT3-2D PDAC model, combination therapy of established (~75mm3) sc tumors reduced tumor growth to a greater extent (PBS: 1349±230 mm3 on day 42 vs B + PD1: 355±161mm3, p<0.0001) following a 28-day treatment program of PBS, B (1mg/kg by daily oral gavage), PD1 (10mg/kg ip weekly) or the combination of B + PD1. Treatment with either B or PD1 alone had minimal anti-tumor effects. B + PD1 therapy was accompanied by significant tumor infiltration of NK cells by IHC, flow cytometry and Nanostring analysis. A dramatic reduction of tumor stromal fibrosis by Masson's trichrome staining was found in tumors treated with B alone or B + PD1. These findings suggest that the combination of BXCL701 with anti-PD1 antibody therapy can exert anti-tumor effects associated with increased intratumoral NK cell content and the loss of fibrosis that may facilitate immunotherapy efficacy. Citation Format: Shangzi Wang, Allison Fitzgerald, Reham Ajina, Sandra A. Jablonsk, Louis M. Weiner, John MacDougall, Veena Agarwal, Vince J. O'Neill. Therapy with BXCL701 (B), a DPP8, DPP9, DPPIV and FAP inhibitor, in combination with anti-PD1 antibody (PD1) in a syngeneic murine pancreatic ductal adenocarcinoma (PDAC) model improves treatment outcomes and induces intratumoral NK cell infiltrates and a marked reduction in tumor stromal fibrosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6636.

Abstract The oral DPP8/9 inhibitor BXCL701 in combination with an anti-PD-1 antibody (aPD-1) [AACR 2017] or in triple combination with aPD-1 and pegylated IL-2 (NKTR-214) [ASCO 2018] has demonstrated inhibition of tumor growth or complete regression respectively in animal models of pancreatic cancer. In the present study, the mechanism of action of BXCL701 has been elucidated in a sub-chronic pharmacokinetic / pharmacodynamic study at molecular and cellular level, by administering BXCL701 as a single agent. BXCL701 (20 μg) was administered orally every day for 14 days in tumor (Pan02) bearing mice. Tumor and serum samples were harvested after 0, 1, 8 and 16 hrs after BXCL701 administration on day 1, 7 and 14. Serum samples were analysed for cytokines and tumor tissues were analysed for infiltrating immune cells and gene expression. BXCL701 is known to inhibit regulatory proteases DPP8/9, consequently activating Nlrp1b inflammasome, which in turn activates pro-caspase-1 to mediate pyroptosis in mouse macrophages [Okondo et al, 2018]. Caspase-1 is involved in cleavage of pro-IL-1β and pro-IL-18 to their active forms, IL-1β and IL-18 respectively [Walle et al, 2016,]. IL-18 was observed to be significantly (p<0.05) upregulated (>50 fold) at 8 hrs on day 1 and achieved steady state levels by day 7 in BXCL701-treated animals. Other cytokines like IL-1β, IFN-γ, G-CSF, IL-5, IL-6, CXCL9, MCP-1, KC and Eotaxin were also observed to be upregulated in BXCL701-treated animals at the 8 hr timepoint on day 1, 7 and 14 in comparison to day 1, 0 hr. BXCL701 significantly upregulates T cells (total T cells, CD4+ T helper cells and CD8+ T cells) infiltration along with NK cells within the tumor microenvironment. It also appears to enhance antigen presentation by upregulating MHC class I genes and MHC class I expressing cells within the tumor. The mechanism of action of BXCL701 was evaluated at the molecular level as well. The comparison of genes in tumor tissues from BXCL701-treated animals vs respective vehicle-treated animals on Day 7 and 14 demonstrated that the upregulated gene clusters were innate and adaptive immune response genes, T-cell receptor genes and MHC genes. Also, genes associated with T cell and NK cell mediated apoptosis and cytolysis e.g. FasL, GzmA, were upregulated and indicates enhanced cell death (e.g. upregulation of pdcd1) within tumor. On the other hand, downregulated gene clusters belonged to functional categories like cell cycle, DNA repair, several genes associated with cancer progression (GPCRs and Olfactory receptors) and extra cellular matrix (ECM) modification (collagen and metalloproteases). In conclusion, BXCL701-treatment induces innate and adaptive immune responses that leads to tumor growth inhibition probably via inducing cell death and reducing ECM modification. Citation Format: Veena Agarwal, John MacDougall, shubhendu Trivedi, Dimple Bhatia, Zeenia Jagga, Hemant Banga, Diane Healy, Sreenivas Adurthi, Vince O'Neill. The dipeptidyl peptidase inhibitor BXCL701 activates innate immunity followed by adaptive immunity on a molecular and cellular level in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 962.

e14253 Background: The oral DPP8/9 and FAP inhibitor BXCL701 in combination with a checkpoint inhibitor anti-PD-1 antibody (aPD-1) has demonstrated inhibition of tumor growth as well as up-regulation of immuno-stimulatory cytokines and tumor-infiltrating immune cells in animal models [ASCO 2018]. In the present study, BXCL701 was evaluated for its potential to improve synergistically the survival of animals when combined with anti-CTLA-4 antibody (aCTLA-4), an immune checkpoint blocker, and galunisertib, a transforming growth factor (TGF) β type I receptor inhibitor that counteracts the immunosuppressive influence that TGF β signalling has in the tumor microenvironment. Methods: In vivo studies were conducted utilizing the syngeneic colon cancer model (MC38) in C57BL/6 mice. BXCL701 was administered at the dose of 20 µg qd, p.o, while aPD-1 and aCTLA-4 were administered at the dose of 5 and 10 mpk q2w, i.p. respectively. Finally, galunisertib was administered at 75 mpk bid. Dosing was carried out for 4 weeks and median survival of the animals was calculated. Results: BXCL701 demonstrated a significant improvement in the survival of animals when combined with aCTLA-4. The median survival of animals treated with combination of BXCL701 and aCTLA-4 was significantly increased to 51.5 days in comparison to 40 days observed in BXCL701-treated animals (p < 0.001). Furthermore, in a separate study, addition of BXCL701 to the double combination of aPD-1 and aCTLA-4 resulted in a median survival of 51 days in comparison to 37 days for aPD-1 and aCTLA-4-treated animals (p < 0.05). Similarly, addition of BXCL701 to the combination treatment of aPD-1 and galunisertib increased the median survival to 40.5 days in comparison to 28 days observed in animals treated with galunisertib and aPD-1 combination (p < 0.05). Conclusions: BXCL701, an innate immune activator, synergistically combines with aCTLA-4 and a combination of aPD-1 with galunisertib to improve survival response likely by turning cold tumors to hot tumors. The present study advocates for a clinical evaluation of BXCL701 in combination with these immunomodulators in solid tumors.

The oral DPP8/9 and FAP inhibitor BXCL701 in combination with an anti-PD-1 antibody (aPD-1) has demonstrated inhibition of tumor growth as well as up-regulation of immuno-stimulatory cytokines and tumor-infiltrating immune cells in animal models [AACR 2017, ASCO 2018]. In the present study, the primary cytokines/chemokines influenced by BXCL701 administered as a single agent have been explored and provide the rational for an efficacy study of BXCL701 in combination with an OX40-agonist antibody (aOX40). Cytokines/chemokines were evaluated in serum samples from animals in an acute PK/PD study revealing that BXCL701 stimulated several pro-inflammatory factors. The most stimulated cytokine was IL-18 which is known to bridge the innate and adaptive immune systems through induction of IFN-γ and the CD134 (OX40) pathway. (J of Immunol, 2006, 177: 234). Therefore, an in vivo study was conducted by combining BXCL701, aOX40 and aPD-1 in a preclinical model (MC38) of colorectal cancer. Evaluation of tumor size on day 23 compared to vehicle control showed aOX40 and BXCL701 treated animals to have tumor growth inhibition (TGI) of 0.8% (p = 0.97), and 8% (p = 0.61) respectively. Importantly, BXCL701 in combination with aOX40 resulted in synergistic inhibition of 51% (p = 0.001) when compared to vehicle. A 43% TGI (p = 0.006) was observed in animals treated with the combination of aPD-1 and aOX40. Addition of BXCL701 to the double combination of aOX40 and aPD-1 resulted into 59% TGI (p = 0.0005). At study end, BXCL701 demonstrated a significant improvement in the survival of animals when combined with aOX40. The median survival of vehicle treated animals was determined to be 30 days. BXCL701 and aOX40-treated animal groups had median survival of 35 days and 29 days, respectively (BXCL701 vs Vehicle p = 0.0958, aOX40 vs Vehicle p = 0.8560). The median survival of animals treated with combination of BXCL701 and aOX40 was significantly increased to 46 days in comparison to BXCL701 alone (p < 0.0001), while combination of aPD1 and aOX40 treated animals had a median survival of 36 days (aPD-1 and aOX40 vs Vehicle p = 0.0991). The triple combination of BXCL701, aOX40 and aPD1 resulted in the median survival of 46 days (p < 0.0001 vs Vehicle). In conclusion, the inclusion of BXCL701 in the drug combinations was necessary for a survival benefit because while the aOX40 and aPD-1 combination showed a significant TGI on day 23, this did not translate into an overall survival benefit. Therefore, BXCL701 contributed to a clear synergistic anti-tumor and survival response within the combinations. Ostensibly, BXCL701-regulated cytokines are responsible for the observed synergism with anOX40 treatment. The present study suggests the clinical evaluation of BXCL701 in combination with an OX40-agonist in solid tumors.Citation Format: Veena R. Agarwal, John MacDougall, Dimple Bhatia, Snigdha Gupta, Zeenia Zagga, Nirmal keshava. Dipeptidyl Peptidase Inhibitor BXCL701 synergizes with an OX40-agonist antibody resulting in synergistic anti-tumor response and survival in an animal model of colorectal cancer by bridging the innate and adaptive arms of the immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-077.

Abstract A new method of targeted photodynamic therapy (PDT) is proposed for potentiating anti-tumor immunity. Human papillomavirus (HPV) virus-like particles (VLPs) bind a broad range of tumor types both, in vitro and in vivo, but do not bind healthy, intact tissues. We have linked a photosensitizer dye, IR700, to the HPV VLPs and applied the dye-coupled particles (IR700-VLP) to tumor cells. When exposed to 690nm light, the bound conjugates induced immediate necrotic-like cell death in ovarian, lung, melanoma and cervical cancer cell lines in vitro, and in melanoma and lung tumors in vivo. We are currently expanding these studies to examine long-term tumor growth/survival and the generation of anti-tumor immunity in C57Bl/6 mice using the TC-1 tumor model, which expresses the HPV oncogenes, E6 and E7. This model allows us to track the T-cell responses generated against the HPV oncogenes, in both primary and metastatic tumor settings. Treatment with IR700-VLP and light exposure induced an influx of CD8+ and CD4+ T-cells into the treated tumors. Ex vivo cultures of whole tumor cell suspensions showed IFN-γ production in PMA/ionomycin-stimulated CD8+ and CD4+ T cells and to a lower extent without stimulation, suggesting that these T-cells may have been primed against the tumor antigens. Our data suggest that local anti-tumor immunity can be induced through the rapid tumor cell death caused by IR700, in conjunction with the ability of HPV VLP to induce an innate immune response.

Abstract 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 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.

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

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.

5058 Background: Initial immunohistochemical analysis revealed that about 50% of ovarian cancer samples expressed sonic Hh ligand in the tumor epithelium, suggesting this signaling pathway might be active in this disease. Thus we examined the activity of IPI-926, a potent inhibitor of the Hh pathway that blocks the activity of Smoothend, in a preclinical model of serous ovarian cancer in an adjuvant and maintenance setting. Methods: Primary human serous ovarian tumor xenograft tissue was utilized to generate tumor explants in NOD/SCID mice. Mice bearing tumors (250-1900 mm3) were randomized into four groups 1) IPI-926 PO gavage q 24 hours along with intraperitoneal (IP) vehicle; 2) paclitaxel and carboplatinum (T/C) IP q 7 days with vehicle by PO gavage q 24 hours; 3) IPI-926 by PO gavage q 24 hours + IP T/C; 4) vehicle by PO gavage q 24 hours + IP vehicle q 7 days. The adjuvant treatment period spanned 19 days. In the maintenance arms of the experiment, T/C treatment was stopped in the two groups receiving T/C. These mice continued to receive vehicle or IPI-926 for 24 days. Tumor volume and mouse weights were regularly assessed in experiments. Mouse and human Gli1 were assessed in tumor explants by RT PCR 24 hours after treatment with IPI-926 or vehicle. Results: In the adjuvant setting, IPI-926 inhibited growth compared to vehicle (p = 0.006). Weight loss was similar for all groups. In the maintenance experiment, continued treatment with IPI-926 further inhibited tumor growth when compared to vehicle. Continued treatment with IPI-926 was required to maintain efficacy. There was a 1,000-fold inhibition of Gli1 expression in the tumor associated stroma when compared to vehicle (p < 0.0002) 24 hours post treatment with IPI-926. This modulation was not seen in the tumor cells. Conclusions: IPI-926 treatment precludes tumor growth in the adjuvant and maintenance therapy setting suggesting the Hh signaling pathway contributes to the pathogenesis of ovarian cancer and may be useful as targeted therapy. The modulation of Gli1 expression in the stromal compartment implies signaling between the tumor and stroma. These data suggest that therapeutic inhibition of the Hh pathway could be an important strategy to extend progression-free survival in ovarian cancer patients. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Infinity Infinity Infinity

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.

Abstract Malignant activation of the Hedgehog (Hh) pathway is associated with multiple tumor types. The Hh pathway can promote growth of certain cancers via three modes of malignant activation: ligand-dependent signaling either to the tumor cell itself or between the tumor and its microenvironment, and ligand-independent signaling activated by genetic mutations. In certain cancers, tumor-derived Hh ligand signals in a paracrine manner to the stromal cells in the microenvironment that support tumor cell growth. Using a sensitive immunohistochemistry method for detecting Hh ligand, 83 percent of 105 human prostate tumor samples, including lymph and bone metastases from the same patients, were found to express high levels of Sonic Hedgehog (SHh) ligand. LuCaP 23.1 and LuCaP 35v xenografts were derived from lymph node metastasis from patients with prostate cancer. Paracrine Hh signaling is detected in both xenograft models, where the human tumor cells produce Hh ligand and the surrounding stromal cells respond with Hh pathway activation, as measured by Gli1 levels. IPI-926, a novel, small molecule Hh inhibitor that acts by blocking Smoothened, inhibits signaling between the tumor and stromal cells in both castration sensitive and castration resistant tumor models. Inhibition of Hh signaling with IPI-926 was then evaluated as a potential means to reduce growth of prostate cancer tumors and metastases. IPI-926 significantly delays relapse and slows the growth rate of castration resistant primary human prostate tumors post-docetaxel treatment. Additional preclinical studies with IPI-926 in combination with chemotherapy and targeted therapies in castration resistant tumor models are in progress. These pre-clinical results provide a rationale for testing Hh inhibitors in patients with castration resistant prostate 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 3857.