Search

Background Chemotherapy is the treatment of choice for metastatic castration-resistant prostate cancer (mCRPC) nonresponsive to androgen receptor–targeted therapies. Nevertheless, the impact of chemotherapy on patient survival is limited and clinical outcome remain dismal. Bromodomain and extraterminal inhibitors (BETis) are attractive therapeutic agents and currently in clinical trials to be tested for their efficacy in prostate cancer patients. Objective In this study, we evaluated the activity of two clinical stage BETis, INCB054329 and INCB057643, alone and in combination with chemotherapeutics used for the treatment of mCRPC. Design, setting, and participants Drug activity was evaluated in vitro by MTT, clonogenic, prostato-sphere, and flow cytometry assays. The activity in vivo was evaluated in mice bearing prostate tumor (22Rv1) xenografts. Outcome measurements and statistical analysis Cell growth data were analyzed to determine the maximum effect and the concentration that reduces by 50%. For concomitant treatments, the combination index was determined according to the Chou-Talalay method. For in vivo activity, changes in tumor size (T/Ci%), weight (T/Cd%), doubling time, and mouse body weight were monitored. Statistical significance was determined by one-way analysis of variance followed by a Student-Newman-Keuls or Turkey a posteriori test. Results and limitations INCB054329 and INCB057643 had significant activity as single agents in human prostate cancer cell lines and 22Rv1 tumor xenografts. Combined treatment with INCB057643 and any of docetaxel, olaparib, or carboplatin was synergistic/additive in vitro. Notably, INCB057643, given with a low-intensity dosing schedule, greatly enhanced the anti-tumor activity of docetaxel, carboplatin, and olaparib in 22Rv1 tumor xenografts. Conclusions Collectively, these results provide the first evidence of the therapeutic benefit obtainable by combining BETis with non–androgen receptor–targeted therapies for the treatment of mCRPC. Patient summary Chemotherapy has limited efficacy in patients with metastatic castration-resistant prostate cancer. This study provides evidence of enhanced efficacy of clinically used chemotherapeutics when given in combination with the bromodomain and extraterminal inhibitor INCB057643, expanding the horizon of the current options for the treatment of prostate cancer.

In pancreatic cancer the tumor microenvironment (TME) can account for up to 90% of the tumor mass. The TME drives essential functions in disease progression, invasion and metastasis. Tumor cells can use epigenetic modulation to evade immune recognition and shape the TME toward an immunosuppressive phenotype. Bromodomain inhibitors are a class of drugs that target BET (bromodomain and extra-terminal) proteins, impairing their ability to bind to acetylated lysines and therefore interfering with transcriptional initiation and elongation. INCB057643 is a new generation, orally bioavailable BET inhibitor that was developed for treating patients with advanced malignancies. KrasG12D/+, Trp53R172H/+, Pdx-1-Cre (KPC) mice mimic human disease, with similar progression and incidence of metastasis. Treatment of established tumors in KPC mice with INCB057643 increased survival by an average of 55 days, compared to the control group. Moreover, INCB057643 reduced metastatic burden in these mice. KPC mice treated with INCB057643, starting at 4 weeks of age, showed beneficial changes in immune cell populations in the pancreas and liver. Similarly, INCB057643 modified immune cell populations in the pancreas of KrasG12D/+, Pdx-1-Cre (KC) mice with pancreatitis, an inflammatory process known to promote pancreatic cancer progression. The data presented here suggest that the bromodomain inhibitor INCB057643 modulates the TME, reducing disease burden in two mouse models of pancreatic cancer. Furthermore, this work suggests that BRD4 may play a role in establishing the TME in the liver, a primary metastatic site for pancreatic cancer.

Objective Homologous recombination (HR)-proficient ovarian tumors have poorer clinical outcomes and show resistance to poly ADP ribose polymerase inhibitors (PARPi). A subset of HR-proficient ovarian tumors show amplification in bromodomain and extra-terminal (BET) genes such as BRD4. We aimed to test the hypothesis that BRD4 inhibition sensitizes ovarian cancer cells to PARPi by reducing HR efficiency and increasing DNA damage. Methods HR-proficient ovarian cancer cell lines (OVCAR-3, OVCAR-4, SKOV-3, UWB1.289+BRCA1) were treated with BRD4-targeting siRNA, novel (INB054329, INCB057643) and established (JQ1) BET inhibitors (BETi) and PARPi (olaparib, rucaparib). Cell growth and viability were assessed by sulforhodamine B assays in vitro, and in SKOV-3 and ovarian cancer patient-derived xenografts in vivo. DNA damage and repair (pH2AX, RAD51 and BRCA1 foci formation, and DRGFP HR reporter activity), apoptosis markers (cleaved PARP, cleaved caspase-3, Bax) and proliferation markers (PCNA, Ki67) were assessed by immunofluorescence and western blot. Results In cultured cells, inhibition of BRD4 by siRNA or INCB054329 reduced expression and function of BRCA1 and RAD51, reduced HR reporter activity, and sensitized the cells to olaparib-induced growth inhibition, DNA damage induction and apoptosis. Synergy was observed between all BETi tested and PARPi. INCB054329 and olaparib also co-operatively inhibited xenograft tumor growth, accompanied by reduced BRCA1 expression and proliferation, and increased apoptosis and DNA damage. Conclusions These results provide strong rationale for using BETi to extend therapeutic efficacy of PARPi to HR-proficient ovarian tumors and could benefit a substantial number of women diagnosed with this devastating disease.

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. TNBC affects younger women and is characterized by earlier rates of relapse, higher frequency of visceral metastases, and shorter survival outcomes when compared to ER+ or HER2+ disease. Although the disease only represents ˜15% of all breast cancer cases, it accounts for 25% of all breast cancer deaths – with treatment options currently limited to chemotherapy. Development of targeted therapies for TNBC is challenging due to molecular heterogeneity and lack of high-frequency “driver” alterations amenable to therapeutic intervention. Recent studies have demonstrated increased sensitivity of TNBC to the anti-proliferative effects of Bromodomain and Extra-Terminal motif inhibitor (BETi) compared to the other breast cancer subtypes. To determine mechanisms of sensitivity to BETi, we analyzed the effect of a BETi across a panel of TNBC cell line models and identified cell lines that were both sensitive and insensitive to BETi. With the intent of identifying biomarkers of sensitivity, we performed RNA-seq and precision nuclear run-on and sequencing (PRO-seq) on both sensitive and insensitive cell line models and data generated identified significant differences in key growth regulatory and apoptotic signaling pathways, including notable differences in Myc-dependent signaling. Our data suggest potential biomarkers of BETi-sensitivity that may be of value in further pre-clinical studies. Further, our results provide mechanistic rationale for combinations of BETi with select, targeted therapies in a disease that is in need of new therapeutic intervention. Citation Format: Schafer JM, Lehmann BD, Redman LN, Liu P, Stubbs M, Ruggeri B, Scherle P, Pietenpol JA. Combination treatment with bromodomain and extra-terminal motif inhibitors in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-24.

Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML. We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints. Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

MCL1 is a member of the anti-apoptotic BCL2 family of proteins and plays a critical role in maintaining cellular homeostasis and promoting cell survival. It is frequently amplified in cancer and increased expression of MCL1 is associated with a higher grade and poor prognosis in multiple tumor types. Importantly, MCL1 has been implicated in mediating resistance to chemotherapy as well as targeted therapies, including the BCL2 inhibitor, venetoclax. Here, we describe the in vitro and in vivo activity of PRT1419, a potent and selective inhibitor of human MCL1, that can induce tumor cell death by apoptosis. PRT1419 inhibits the binding of MCL1 to its physiological ligand, BIM, with low nanomolar potency. PRT1419 also demonstrated >200-fold selectivity against other BCL2 family members, including BCL2 and BCL-XL. In vitro, PRT1419 treatment resulted in robust activation of apoptotic markers such as cleaved caspase-3 in a concentration-dependent manner in several cancer cell lines. Consistent with its pro-apoptotic effects, PRT1419 treatment led to robust inhibition of cell proliferation in a concentration-dependent manner in a panel of cancer cell lines. Cell lines representing hematologic cancers as well as a subset of breast and non-small cell lung cancer lines were sensitive to PRT1419, and this response was associated with a significantly higher MCL1/BCL-XL mRNA ratio. Also, PRT1419 treatment resulted in potent, concentration-dependent cytotoxic activity ex vivo in patient-derived xenograft (PDX) models of various subtypes of human sarcoma, breast and esophageal cancer. PRT1419 demonstrated good oral bioavailability and favorable pharmacokinetic properties in vivo. In subcutaneous cell-line derived xenograft (CDX) models of multiple myeloma, acute myeloid leukemia (AML) and diffuse large B-cell lymphoma, oral administration of PRT1419 demonstrated potent anti-tumor activity with complete tumor regressions observed at tolerable doses. This response correlated with a dose-dependent induction of cleaved caspase-3 and cleaved-PARP in tumor tissue. Significant in vivo activity, including complete responses, was also observed in PDX models of lymphoma. In preclinical models of solid tumors, PRT1419 demonstrated significant tumor growth inhibition in a PDX model of human soft tissue sarcoma and a CDX model of breast cancer. PRT1419 was also tested in combination with other approved targeted therapies in vitro and in vivo. In AML, combining PRT1419 with a BCL2 inhibitor revealed a synergistic interaction in cell lines, ex vivo PDX models as well as a CDX model in vivo. Further, PRT1419 demonstrated synergistic activity with tyrosine kinase inhibitors to inhibit the proliferation of breast, melanoma, and non-small cell lung cancer cell lines. PRT1419 is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies (NCT04543305). Citation Format: Neha Bhagwat, Alexander Grego, William Gowen-MacDonald, Min Wang, Miles Cowart, Xiaowei Wu, Jincong Zhuo, Andrew Combs, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Preclinical characterization of PRT1419, a potent, selective and orally available inhibitor of MCL1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 983.

Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMT that catalyzes the formation of symmetrical dimethyl arginine (SDMA) on protein substrates and plays important roles in regulating RNA splicing as well as transcription and activity of critical oncogenic signaling pathways. Recent studies have revealed PRMT5 as a potential therapeutic target for various cancers including Central Nervous System (CNS) tumors such as glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. Several PMRT5 inhibitors have entered into the clinic for hematological and solid tumors, but none have been reported to be brain penetrant, a potentially desirable feature of a small molecule designed for treating CNS cancers and brain metastases. PRT811 is a potent, selective, and brain penetrant PRMT5 inhibitor, and exhibits potent in vitro and in vivo activities in preclinical models of brain tumors. PRT811 inhibited the methyltransferase activity of the PRMT5/MEP50 complex with an IC50 of 3.9 nM, but showed minimal inhibition against a panel of 37 human methyltransferases at 10 µM. In a panel of brain cancer cell lines of varied histology, PRT811 reduced SDMA levels and inhibited cell proliferation with IC50 values in the range of 7-40 nM and 29-134 nM, respectively, regardless of expression levels of methylguanine methyltransferase (MGMT), the expression of which is associated with temozolomide (TMZ) resistance. Furthermore, PRT811 demonstrated broad antiproliferative activity in a panel of 87 cell lines consisting of brain, breast, lung, and skin cancers, representing primary and the most common cancer types that metastasize to the brain. Finally, PRT811 effectively inhibited cell growth in a panel of patient derived xenograft models of GBM in ex vivo 3D cultures. PRT811 is a highly permeable molecule with high brain penetrance in rodents, with an efflux ratio PRT811 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors, gliomas, and myelofibrosis (NCT04089449). Citation Format: Yang Zhang, Hong Lin, Min Wang, Dimitrios Angelis, Michael Hawkins, Dave Rominger, Tom Emm, Juan Luengo, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Discovery of PRT811, a potent, selective, and orally bioavailable brain penetrant PRMT5 Inhibitor for the treatment of brain tumors [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 2919.

Acute myelogenous leukemia (AML) is the most common acute leukemia in adults. A hematologic cancer, the disease is highly heterogeneous, with multiple subtypes. Despite advances in treatment, the long-term survival for AML remains poor and the development of novel treatments is an unmet need. Due to the highly divergent subtypes and mutation profiles in AML, the use of patient-derived models may improve drug discovery and development. To address this, we have established a short-term culture system that supports the growth of primary AML cells ex vivo to permit the evaluation and/or screening of candidate therapeutic agents. Our AML bank is comprised of patient-derived specimens across a range of subtypes (which includes M1, M2, M4, M5, NOS and others), and includes models with common mutations in FLT3 (ITD), IDH1/IDH2 and NPM. Primary AML specimens were characterized for common mutations by TruSight sequencing and for surface marker expression by flow cytometry. The ex vivo assay system was evaluated for the ability to support the survival and expansion of primary AML specimens. Among these, the majority of the models showed evidence of proliferation, a few models had no net expansion, and some failed to survive. Extension of the culture period for up to 14 days was feasible, with most models having equal or increased cell numbers by the end of the culture period (8 of 10 models evaluated). All models stably expressed CD33 throughout the assay. To verify the applicability of this system for drug testing, a standard of care agent cytarabine (Ara-C) was assessed for each of the AML models. A small cohort of models with distinct mutations were tested for sensitivity towards small molecule inhibitors venetoclax, gilteritnib, glasdegib, panobinostat and Ibrutinib. Cell growth/viability was assessed using Cell titer-Glo assay. Concentration-dependent responses to Ara-C were observed across multiple models (IC50 10 nM to 150 nM), indicating a range of relatively sensitive to resistant AML models. Heterogeneous concentration-dependent responses were observed across multiple patient-derived ex vivo models when treated with venetoclax, glasdegib, gilteritnib, ibrutinib and panobinostat, with both sensitive and resistant models identified. A cohort of models were evaluated in vivo for sensitivity to Ara-C. Systemic engraftment of the patient-derived xenograft AML models (into NOG or NOG-EXL mice) was evaluated by flow cytometry. When engrafted was confirmed, AML-bearing mice were randomized into Ara-C or vehicle control groups. Two weeks later, mice were evaluated for the presence of human CD45+CD33+AML cells. Models that were relatively sensitive to Ara C ex vivo (IC50 < 30 nM) generally showed a greater in vivo response as evidenced by a significant reduction in the mean circulating AML cells versus models with IC50 values >100 nM that had no response to Ara C in vivo. Over thirty distinct patient-derived primary, unpassaged models of AML have been studied both in ex vivo culture and as in vivo systemic models. The diversity in these AML models is reflective of patient diversity, enhancing their utility in the evaluation of novel therapeutic candidates. These data indicate the feasibility of utilization of these primary models, ex vivo as well as in vivo, for drug discovery for AML, from screening to preclinical efficacy modeling. Disclosures Verma: Champions Oncology: Employment. Biel:Champions Oncology: Employment. Ruggeri:Champions Oncology: Consultancy. Wesa:Champions Oncology: Employment, Other: stock options.

Acute myelogenous leukemia (AML) is the most common acute leukemia in adults. A hematologic cancer, the disease is highly heterogeneous, with multiple subtypes. Despite advances in treatment, the long-term survival for AML remains poor and the development of novel treatments is an unmet need. Due to the highly divergent subtypes and mutation profiles in AML, the use of patient-derived models may improve drug discovery and development. To address this, we have established a short-term culture system that supports the growth of primary AML cells ex vivo to permit the evaluation and/or screening of candidate agents. Our AML bank is comprised of patient-derived specimens across a range of subtypes (which includes M1, M2, M4, M5, and others), and includes models with common mutations in FLT3 (ITD), IDH1/IDH2 and NPM. Primary AML specimens were characterized for common mutations by TruSight sequencing and for surface marker expression by flow cytometry. The ex vivo assay system was evaluated for the ability to support the survival and expansion of 22 primary AML specimens. Among these, 15 had evidence of proliferation, 5 had no net expansion, and 2 failed to survive. Extension of the culture period for up to 14 days was feasible, with most models having equal or increased cell numbers by the end of the culture period (8 of 10 evaluated). All models stably expressed CD33 throughout the assay. To verify the applicability of this system for drug testing, a standard of care agent cytarabine (ara-C) was assessed for each of the AML models. Cell growth/viability was assessed using Celltiter-Glo assay. Concentration-dependent responses to ara-C were observed across multiple models (IC50 10 nM to 150 nM), indicating a range of relatively sensitive to resistant AML models. A cohort of models were evaluated in vivo for sensitivity to ara-C. Engraftment of the systemic patient-derived xenograft AML models (into NOG or NOG-EXL mice) was evaluated in circulation by flow cytometry. When engrafted, AML-bearing mice were randomized into ara-C or vehicle control groups. Two weeks later, mice were evaluated for the presence of human CD45+CD33+ AML cells. Models that were relatively sensitive to Ara C ex vivo (IC50 < 30 nM) showed a greater in vivo response as evidenced by a 60-80% reduction in the mean circulating AML cells versus models with >100 nM IC50 values, that had little response. In conclusion, Champions offers over 30 well characterized models of AML for study in ex vivo culture and in vivo systemic xenograft models. The diversity in these AML models is reflective of patient diversity, enhancing their utility in the evaluation of novel therapeutic candidates. These data indicate the feasibility of utilization of these primary models, ex vivo as well as in vivo, for drug discovery for AML, from screening to preclinical efficacy modeling. Citation Format: Bhavna Verma, Swetha Tati, Bruce Ruggeri, Amy Wesa. Establishment of a patient-derived acute myeloid leukemia (AML) ex vivo platform for evaluation of novel therapeutic agents [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 3710.

Current therapies for late-stage systemic lupus erythematosus (SLE) are limited to cytotoxic agents. Delanzomib (CEP-18770) is an orally active, reversible P2 threonine boronic acid inhibitor of the 26S mammalian proteasome. Delanzomib was tested in a head-to-head comparison against bortezomib to protect and treat mice with fatal lupus nephritis (LN). Age matched MRL/lpr or NZBWF1 mice with established SLE or LN, respectively, were treated with delanzomib either 3 mg/kg once or twice weekly intravenously or orally at 10 mg/kg. Mice were also treated with reference agent bortezomib at 0.5 mg/kg, intraperitoneally, once a week or 0.3 mg/kg once or twice a week. Reductions in the frequencies of specific anti-chromatin, smith and dsDNA antibody secreting cells and levels of the corresponding circulating antinuclear antibodies, were observed following delanzomib treatment. Reductions in several serum pro-inflammatory cytokines were observed in delanzomib-treated animals. Delanzomib treatment suppressed the development and progression of renal tissue damage and extended the survival of ill mice. Proteinuria was significantly decreased and severity of various renal histopathologies reduced relative to vehicle-treated nephritic mice. Treatment of lupus in these models demonstrated that delanzomib treatment lead to greater tolerability and rate of response resulting in improved stabilization of disease.

Introduction: Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (REACH1: NCT02953678; REACH2: NCT02913261). Methods: Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells received from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the potent and selective JAK1/JAK2 inhibitor, ruxolitinib (60 mg/kg) twice daily. Engraftment was analyzed for the proportion of donor and host leukocytes (CD45, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analyses. Colon samples were sectioned at approximately 5 microns, and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3/STAT5, CD3+phosphoSTAT3/STAT5 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of ruxolitinib in corticosteroid refractory mice were evaluated by treating disease bearing BALB/c mice twice daily with an optimal oral dose of prednisolone (1 mg/kg) until the GvHD scores were comparable to vehicle treated mice. Steroid resistant animals were switched to ruxolitinib to determine the potential efficacy benefit in comparison to mice maintained on ruxolitinib treatment alone for the duration of the study. Results: Oral ruxolitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss, improving GvHD scores and had no detrimental effects on donor engraftment. Comparable efficacy was observed between prophylactic and therapeutic treatment with 60 mg/kg twice daily dosing regimens. Oral ruxolitinib administration in BALB/c mice achieved JAK1/JAK2 IC50 coverage for 8 h with 60 mg/kg twice daily. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Ruxolitinib (60 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen in groups treated with ruxolitinib, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with JAK/STAT pathway inhibition as measured by significant reductions in normalized phosphoSTAT3/STAT5 in T cells and colonic epithelial cells. Prednisolone was transiently effective until day 31 in reducing GvHD scores. Switching treatment regimens from prednisolone to ruxolitinib twice daily rapidly and significantly enhanced GvHD efficacy to a level comparable to ruxolitinib monotherapy. In a dose cessation and resumption paradigm, ruxolitinib restored efficacy when treatment was resumed on day 33 after being halted on day 24. In addition, ruxolitinib significantly enhanced the survival of the recipient BALB/c mice in comparison to vehicle treated animals. Conclusions: Ruxolitinib, a JAK1/JAK2 selective inhibitor, significantly ameliorated GvHD severity with no detrimental effects on engraftment in both a steroid untreated and a corticosteroid refractory MHC-mismatched model of acute GvHD. Efficacy was associated with JAK/STAT pathway inhibition in colon and target tissue infiltrating T-cells. In a dose cessation and resumption paradigm, ruxolitinib restored efficacy once treatment was resumed. In addition, ruxolitinib reduced disease burden and enhanced survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. Disclosures Juvekar: Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

Introduction: Graft-versus-host disease (GvHD) is a severe complication arising in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Potent and selective modulation of JAK1/STAT-mediated signaling is an attractive therapeutic strategy for the management of acute GvHD and is currently being evaluated in clinical trials (GRAVITAS-301: NCT03139604; GRAVITAS-119: NCT03320642). Methods: Acute GvHD was induced in BALB/c mice using the established MHC-mismatched mouse model. BALB/c (H-2Kd) recipients were given an intravenous injection of a combination of splenocytes and T cell depleted bone marrow cells from allogeneic cell transfer from donor C57BL/6 (H-2Kb) mice. Animals were dosed orally with vehicle or the selective JAK1 inhibitor, itacitinib (60 mg/kg or 120 mg/kg twice daily). Engraftment was analyzed for the proportion of donor and host leukocytes (CD45+, H-2Kb, and H-2Kd). GvHD clinical scores were assessed by standard methods and inflammatory cytokine profiles in blood and colon quantified by multiplex analysis. Colon samples were sectioned and stained with the following immunohistochemical (IHC) markers: CD4, CD8, phosphoSTAT3 and CD3+phosphoSTAT3 (dual staining) for pharmacodynamic assessment of JAK/STAT pathway activity in colon and infiltrating T-cells. Effects of itacitinib on preservation of Graft-versus-Leukemia (GVL) were evaluated by injecting BALB/c mice with A20 lymphoma cells that are of H-2Kd phenotype along with combination of splenocytes and T cell depleted bone marrow from C57BL/6 (H-2Kb) mice. Results: Itacitinib administration was highly effective in both prophylactic (from day −3) and therapeutic (from day 14) dosing regimens in ameliorating body weight loss and improving GvHD scores. Itacitinib did not significantly impact donor engraftment as determined by CD45+/H-2Kb quantification by flow cytometry. Similar efficacy was observed with 60 mg/kg versus 120 mg/kg twice daily dosing regimens. Oral itacitinib administration achieved JAK1 IC50 coverage for 4 h and 12 h at 60 mg/kg twice daily and 120 mg/kg twice daily, respectively. Associated with GvHD progression, maximal upregulation of inflammatory cytokines were observed in peripheral blood on day 17 (IFN-γ, TNF-α, IL-6, IL-13) and in colon on day 28 (IFN-γ, TNF-α, IL-1β). Itacitinib (120 mg/kg twice daily) treatment significantly reduced the inflammatory cytokine milieu at these disease stages. No differences were observed in absolute number of CD4+ T cells and CD8+ T cells in blood and spleen with itacitinib treatment, but significant reductions were detected in CD4+ T cells and CD8+ T cells in the inflamed colon tissue along with significant JAK1/STAT3 inhibition as measured by reductions in normalized pSTAT3 in T cells and colonic epithelial cells. Itacitinib treatment did not negatively impact GVL responses, as evidence by T cell mediated reduction of tumor burden. Furthermore, itacitinib treatment enhanced the survival of the recipient BALB/c mice in comparison to the vehicle treated animals. Conclusions: Itacitinib, a selective JAK1 inhibitor ameliorated GvHD severity when administered prophylactically or therapeutically and had no detrimental effects on engraftment and preservation of GVL. Furthermore, itacitinib inhibited JAK1/STAT3 activation in diseased colon tissue and infiltrating T-cells, and reduced disease burden and improved survival by modulating levels of inflammatory cytokines important in the pathophysiology of acute GvHD. Disclosures Juvekar: Incyte Corporation: Employment. Ruggeri:Incyte Corporation: Employment. Condon:Incyte Corporation: Employment. Borkowski:Biomodels LLC: Employment. Huber:Incyte Corporation: Employment. Smith:Incyte Corporation: Employment.

Non-small cell lung cancers (NSCLC) make up the majority of lung cancers, and are predominantly driven by aberrant kinase pathway signaling. Oncogenic mutations leading to activation of the epidermal growth factor receptor (EGFR) have been identified in a substantial fraction of NSCLC patients, leading to EGFR-targeted therapies such as erlotinib that have improved patient outcome. However, inhibition of EGFR consistently leads to drug resistance through multiple pathways, creating a therapeutic need in NSCLC. One particular route to resistance of EGFR inhibitors is activation of pathways that can bypass the need for signaling through the EGFR, such as the JAK/STAT pathway. To explore the impact of JAK/STAT pathway modulation on EGFR inhibitor resistance, combination efficacy studies evaluating the JAK1 selective inhibitor itacitinib with either erlotinib or the EGFR T790M mutant inhibitor osimertinib, were conducted in xenograft models of activated and erlotinib resistant NSCLC. The HCC827 xenograft (EGFR-activating deletion in exon 19) model was very sensitive to both erlotinib and osimertinib, while the NCI-H1975 xenograft (EGFR T790M/L858R) model responded only to osimertinib. Itacitinib was efficacious in the HCC827 model, while only marginal tumor growth inhibition was observed with itacitinib in the NCI-H1975 model despite both models having detectable levels of pSTAT3. The combination of itacitinib with either erlotinib or osimertinib inhibited tumor growth to a greater degree than monotherapies in the HCC827 model. Despite marginal single agent efficacy from itacitinib in the NCI-H1975 model, itacitinib enhanced the efficacy of osimertinib at several dose levels in this model. Importantly, itacitinib and erlotinib administration had synergistic efficacy in this erlotinib-resistant model, indicating that JAK1 specific signaling may be a critical bypass mechanism for resistance to EGFR inhibitors. Downstream of EGFR, both erlotinib and osimertinib inhibited different signaling pathways when combined with itacitinib in the NCI-H1975 model: STAT signaling was regulated by erlotinib, while the AKT/S6 and ERK pathways were regulated by osimertinib. An analysis of possible upstream activators of signaling pathways relevant to NSCLC survival revealed that IL-6, MCP-1 and IL-8 levels were altered in H1975 tumors from mice treated with the combination of itacitinib and osimertinib, and to a lesser extent with the combination of itacitinib and erlotinib. These data demonstrate the potential utility of the JAK1 specific inhibitor itacitinib in EGFR activated NSCLC, or for patients with EGFR mutations who are no longer responsive to a first generation EGFR inhibitor such as erlotinib. The combination of itacitinib and osimertinib is currently in a Phase I/II study (NCT02917993). Citation Format: Matthew C. Stubbs, Xiaoming Wen, Chu-Biao Xue, Taisheng Huang, Wenqing Yao, Brian Metcalf, Reid Huber, Peggy Scherle, Bruce Ruggeri. In vivo assessment of the combination of the JAK1 selective inhibitor itacitinib with first- and second-generation EGFR inhibitors in models of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2938.

The primary cause of cancer associated mortality is tumor metastasis. The concept of the tumor pre-metastatic niche is supported by evidence of changes at distal pre-metastatic sites that create a permissive environment to allow disseminated tumor cells to seed. Myeloid-derived suppressor cells (MDSCs) remodel the tumor microenvironment and function as immunosuppressive cells to promote tumor growth. Previously, we demonstrated that the clinical stage LSD1 specific inhibitor, INCB059872 significantly reshaped the myeloid compartment in the murine 4T1 syngeneic murine model of breast cancer. Treatment with INCB059872 significantly reduced the population of MDSCs in the tumor microenvironment. Since it has been reported that MDSCs promote establishment of a pre-metastatic niche, we hypothesized that INCB059872 could suppress or delay metastatic processes in the 4T1 model and thereby could impact spontaneous metastases to the lung. In vitro, INCB059872 significantly suppressed cancer cell migration of triple negative breast cancer cells, SUM145PT. In vivo, the effect of INCB059872 on forming the metastatic niche using the 4T1 mouse breast tumor model was explored. Vehicle treated animals exhibited a significant infiltration of MDSCs to the primary tumor and lungs prior to cancer cells metastasizing. In contrast, INCB059872 administration significantly suppressed the infiltration of MDSCs in primary tumor and lung tissues. Histological analyses further demonstrated the reduction of metastatic loci in lung with INCB059872 treatment. Plasma levels of CCL2, a cytokine which is required for the recruitment and functional specialization of MDSCs, were significantly reduced in animals treated with INCB059872. These data suggest a possible mechanism to reduce infiltration of MDSCs into lung tissues. Notably, analyses of molecular pathways using RNA-Seq identified that components of the EMT associated pathway are also downregulated in tumors treated with INCB059872, which further supports the role of INCB059872 in the inhibition of metastasis. Taken together, these preclinical data suggest that inhibition of LSD1 with INCB059872 can suppress metastasis through multiple molecular and cellular mechanisms, notably by inhibition of the formation of the pre-metastatic niche by modulating the population of MDSCs in the primary tumor and distal tissues. Citation Format: Sang Hyun Lee, Melody Diamond, Antony Chadderton, Huiqing Liu, Alla Volgina, Valerie Roman, Michael Weber, Chunhong He, Rebecca Stewart, Denise Hertel, Phillip Liu, Liangxing Wu, Julian Oliver, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. The FAD-directed LSD1 specific inhibitor, INCB059872, inhibits cell migration and metastasis by suppressing premetastatic niche formation in a spontaneous metastasis mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3929.

Acute myeloid leukemia (AML) is characterized by infiltration of abnormally differentiated, clonal and highly proliferative cells of the hematopoietic system that acquire successive genomic alterations. AML is the most common acute leukemia in adults. Current therapies are of limited utility and involve a combination of cytarabine and anthracycline based regimens with allogeneic stem cell transplantation for eligible candidates, but there is an urgent need to improve therapies for AML. JAK/STAT pathway dysregulation plays a role in the pathogenesis of AML and the JAK2 V617F mutation is present in only a small percentage of these patients. Studies were conducted to evaluate the in vitro and in vivo activities of INCB052793, a highly JAK1-selective inhibitor having 100-fold selectivity for JAK1 over JAK2 in cell lines, xenograft and PDX models of human AML having elevated endogenous pSTAT3 and or pSTAT5 activation. In vitro, INCB052793 effectively inhibited p-Stat3 and/or p-Stat5 phosphorylation MV411, Molm 16 and Molm 13 cell lines and caused marked reductions in p-Akt and c-Myc levels in MV411 and Molm16 cells. Given these observations, oral administration of INCB052793 was evaluated at doses of 3-30 mg/kg twice daily in MOLM-16 xenografts and FLT3-ITD AML xenograft models, MV-4-11 and Molm-13, in SCID mice. INCB052793 administration significantly inhibited tumor growth in MOLM-16 xenografts in a dose dependent manner and resulted in complete downregulation of p-Stat3 and p-Stat5 levels in MOLM-16 tumors. Similarly, INCB052793 administration was highly effective in inhibiting tumor growth in FLT3-ITD AML models, MV-4-11 and Molm-13. Administration of INCB052793 in a systemic PDX model of AML with elevated endogenous levels of p-Stat3 and p-Stat5 resulted in amelioration of disease severity and demonstrated a significant effect on median survival in leukemic SCID mice. All dosing regimens of INCB052793 in both xenograft and PDX models were well tolerated. Since azacitidine and cytarabine are standards of care for the treatment of AML, the efficacy of INCB052793 was benchmarked against optimal dosing regimens of these agents. In the AML xenograft models evaluated, INCB052793 was comparably or more efficacious in reducing tumor burden than azacitidine and cytarabine. The combination of INCB052793 with cytarabine showed superior efficacy in comparison to single agents in the MOLM-16 xenograft model, and combinatorial studies are in progress in additional AML models. These findings suggest the therapeutic potential of INCB052793 as a single agent and in combination with standard of care chemotherapeutic regimens for the management of AML. Citation Format: Ashish Juvekar, Sindy Condon, Xiaoming Wen, Bruce Ruggeri, Peggy Scherle, Reid Huber, Yunlong Li, Wenqing Yao, Song Mei, Deepak Bhasin, Maria Mancini. INCB052793, a JAK1 selective inhibitor, is highly efficacious in PDX and xenograft models of acute myeloid leukemia (AML) expressing elevated endogenous pSTAT3/pSTAT5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1876.

Castration-resistant prostate cancer (CRPC) is an advanced stage of the disease for which there are limited treatment options. Multiple genetic and epigenetic events contribute to the emergence of CRPC. Bromodomain and extra-terminal (BET) proteins are attracting considerable attention as targets for prostate cancer therapy due to their regulatory role and impact on multiple genes involved in tumor progression and treatment resistance. Several BET bromodomain inhibitors are currently in clinical trials for cancer treatment. In this study, we evaluated the efficacy of the BET inhibitor INCB057643, which is currently in phase 2 clinical trials, as single agent and in combination with enzalutamide or docetaxel in prostate cancer models. The anti-proliferative activity and the effects of INCB057643 on colony and tumor-sphere forming capacity were evaluated in vitro in androgen-dependent (LNCaP and VCaP) and androgen-independent (DU145, PC3, 22Rv1) cells. The effect of the combination of INCB057643 with enzalutamide or docetaxel on cell growth was evaluated with MTT or SRB methods. The in vivo efficacy of INCB057643 as single agent and in combination was assessed in 22Rv1 mouse xenografts. INCB057643 showed significant anti-proliferative activity in all the prostate cancer cell lines. Interestingly, INCB057643 exhibited substantially higher activity in colony and tumor-sphere forming assays in all cell lines. This was particularly evident in 22Rv1 cells, suggesting a strong impact on tumorigenic stem-like cell subpopulation in this CRPC cell model. The combination of INCB057643 with docexatel was additive or synergistic in DU145, 22Rv1 and LNCaP cells (CI of 0.46, 1.04 and 0.66, respectively). Also, concomitant and sequential treatment with INCB057643 and enzalutamide resulted in potentiation of the antiproliferative effect in 22Rv1 and LNCaP cells. These results were mirrored in 22Rv1 tumor xenografts, where the INCB057643/docetaxel and INCB057643/enzalutamide combinations resulted in potentiation and significant reduction of tumor growth compared to control and/or single agent-treated mice. In summary, INCB057643 has significant activity both in vitro and in vivo and enhances the antitumor effect of both docetaxel and enzalutamide in 22Rv1 cells, a model of CRPC. These results point to INCB057643 as promising agent for treatment of CRPC and development of novel drug combination strategies. Citation Format: Ramiro Vázquez, Gianluca Civenni, Giada Zoppi, Dheeraj Shinde, Aleksandra Kokanovic, Phillip Liu, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. Anti-tumor efficacy of INCB057643, a novel BET bromodomain inhibitor, in castration-resistant prostate cancer as single agent and in combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5793.

Despite the successes of immune checkpoint blockade for the treatment of a variety of cancers, effective combinatorial therapy strategies are needed to achieve more durable and complete clinical responses in patients. Pharmacologically inducing a more permissive tumor microenvironment to enhance patient responsiveness to immune modulatory therapies may offer a rational approach to address this medical need. In particular, targeting immune suppressive myeloid cells, including myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), and polymorphonuclear (PMN) cells in the tumor microenvironment may enhance the effectiveness of immune checkpoint blockade. Since MDSCs abundantly infiltrate syngeneic 4T1 mammary tumors compared with other commonly used syngeneic tumor models, this model was chosen for testing the hypothesis that the modulation of MDSC activity enhances antitumor activity driven by adaptive immunity. We have recently demonstrated (#4635, AACR 2017) that the selective Lysine Specific Demethylase 1 (LSD1) inhibitor, INCB059872, redirected myeloid differentiation toward monocyte/macrophages in vitro and in vivo and reduced the PMN-MDSC population in the syngeneic 4T1 murine mammary tumor model. The combination of INCB059872 and PD1/PDL1 axis blockade enhanced antitumor activity and was well tolerated in this model. In this study, we further tested if modulation of MDSCs with INCB059872 could enhance the effect of mechanistically distinct immunotherapeutic agents. The combination of INCB059872 with agonist anti-OX40 or anti-GITR T cell costimulatory monoclonal antibodies significantly augmented antitumor efficacy in the 4T1 model. These results consistently demonstrated that the inhibition of immune suppressive MDSCs increased antitumor activity of immune checkpoint modulatory monoclonal antibodies. Next, we tested the combination of INCB059872 with small-molecule inhibitors targeting the tumor microenvironment. The combination of INCB059872 with highly selective small-molecule immunotherapeutic inhibitors such as epacadostat (IDO1 inhibitor) and ruxolitinib (JAK1/JAK2 inhibitor) demonstrated similar marked increases in antitumor efficacy. The studies to understand mechanism of enhanced activity are under way. In summary, consistent with previous findings, the combination of INCB059872 with a variety of mechanistically distinct immunotherapeutic agents significantly enhanced antitumor efficacy in the syngeneic 4T1 murine mammary tumor model. These results strongly support the hypothesis that reshaping the tumor microenvironment by redirecting myeloid differentiation as a result of LSD1 inhibition enhances the responsiveness of the tumor microenvironment to immunotherapies, supporting the therapeutic rationale for the combination of an LSD1 inhibitor with various immunotherapeutic agents to improve clinical responses in cancer patients. Citation Format: Sang Hyun Lee, Melody Diamond, Antony Chadderton, Thomas Condamine, Huiqing Liu, Valerie Roman, Jin Lu, Yan Zhang, Maxim Soloviev, Chunhong He, Liangxing Wu, Holly Koblish, Timothy Burn, Andrew Combs, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. The LSD1 specific inhibitor INCB059872 enhances the activity of mechanistically distinct immunotherapeutic agents in the syngeneic 4T1 mouse mammary tumor model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A168.

// Fabrizio Tabbo 1,2 , Marco Pizzi 2,3 , Peter W. Kyriakides 2 , Bruce Ruggeri 4 and Giorgio Inghirami 1,2,5 1 Department of Molecular Biotechnology and Health Science and Center for Experimental Research and Medical Studies (CeRMS), University of Torino, Torino, Italy 2 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY, USA 3 General Pathology and Cytopathology Unit, Department of Medicine-DIMED, University of Padova, Padova, Italy 4 Pre-Clinical Discovery Biology, Incyte Corporation, Wilmington, DE, USA 5 Department of Pathology, and NYU Cancer Center, New York University School of Medicine, New York, NY, USA Correspondence to: Giorgio Inghirami, email: // Keywords : tyrosine kinase fusions, translocations, signaling pathways, small molecule inhibitors, resistance mechanisms Received : October 14, 2015 Accepted : January 24, 2016 Published : March 02, 2016 Abstract Cancer biology relies on intrinsic and extrinsic deregulated pathways, involving a plethora of intra-cellular and extra-cellular components. Tyrosine kinases are frequently deregulated genes, whose aberrant expression is often caused by major cytogenetic events (e.g. chromosomal translocations). The resulting tyrosine kinase fusions (TKFs) prompt the activation of oncogenic pathways, determining the biological and clinical features of the associated tumors. First reported half a century ago, oncogenic TKFs are now found in a large series of hematologic and solid tumors. The molecular basis of TKFs has been thoroughly investigated and tailored therapies against recurrent TKFs have recently been developed. This review illustrates the biology of oncogenic TKFs and their role in solid as well as hematological malignancies. We also address the therapeutic implications of TKFs and the many open issues concerning their clinical impact.

Brain injury from inorganic Pb(2+) is considered the most important environmental childhood health hazard worldwide. The microvasculature of the developing brain is uniquely susceptible to high level Pb(2+) toxicity (ie, Pb(2+) encephalopathy) characterized by cerebellar hemorrhage, increased blood-brain barrier permeability, and vasogenic edema. However, the specific molecular mediators of Pb(2+) encephalopathy have been elusive. We found that Pb(2+) induces vascular endothelial growth factor/vascular permeability factor (VEGF) in cultured astrocytes (J Biol Chem, 2000;275:27874-27882). The study presented here asks if VEGF dysregulation contributes mechanistically to Pb(2+) encephalopathy. Neonatal rats exposed to 4% Pb-carbonate develop the histopathological features of Pb(2+) encephalopathy seen in children. Cerebellar VEGF expression increased approximately twofold (p < 0.01) concurrent with the development of cerebellar microvascular hemorrhage, enhanced vascular permeability to serum albumin, and vasogenic cerebellar edema (p < 0.01). No change in VEGF expression occurred in cerebral cortex that does not develop these histopathological complications of acute Pb(2+) intoxication. Pb(2+) exposure increased phosphorylation of cerebellar Flk-1 VEGF receptors and the Flk-1 inhibitor CEP-3967 completely blocked cerebellar edema formation without affecting microhemorrhage formation or blood-brain barrier permeability. This establishes that Pb(2+)-induced vasogenic edema formation develops via a Flk-1-dependent mechanism and suggests that the vascular permeability caused by Pb(2+) is Flk-1 independent.

Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk disease with a frequency of about 20% in patients of all ages. Ph-like ALL can be characterized in different groups based on their chromosomal rearrangements. The most common rearrangements involve CRLF2, ABL1, and EPOR and lead to activation of JAK kinase dependent activation of STAT5, PI3 kinase and other signaling pathways. We have previously shown that Ph-like ALL is a high-risk disease with a poor prognosis in older adults. The focus of this study is to further understand the biology of Ph-like ALL and how the cells develop drug resistance by modulating their signaling pathways to escape apoptosis. Results: We and others have identified activated STAT5 and AKT signaling in Ph-like ALL cell lines and patient samples. Interestingly, inhibition of the JAK-STAT signaling pathway using JAK-kinase inhibitors abrogated short term STA5 signaling as well as AKT signaling, but Ph-like ALL cells showed only a mild apoptotic effect or arrest in cell proliferation in vitro. Furthermore, even though xenotransplantation models of Ph-like ALL show a decrease in leukemia burden when treated with ruxolitinib, a JAK1/2 inhibitor, all mice ultimately succumb to the disease, demonstrating that Ph-like ALL does not demonstrate oncogene addiction. Based on these results we hypothesize that Ph-like ALL cells can adapt to JAK kinase inhibition by activating other signaling pathways. To test this hypothesis, we performed a time course experiment in Ph-like ALL cell lines and detected AKT reactivation shortly after JAK-kinase inhibition. In addition, we detected activation of SYK and a reduction in the PI3K negative regulator PTPN6. These observations lead to the hypothesis that Ph-like ALL may have expression and activation of the pre-B cell receptor (pre-BCR). Indeed, gene expression data from the clinical trial P9906 demonstrate that Ph-like ALL cases with CRLF2-rearrangment and JAK2 mutations express high mRNA levels of pre-B cell receptor signaling molecules, including BLNK, IGGL1, VpreB3, and ZAP70. We next hypothesized that pre-BCR mediated activation of PI3 kinase rescues Ph-like ALL cells from JAK inhibition. To study PI3K signaling we first treated Ph-like ALL cell lines and xenografts with idelalisib or INCB-50465, both selective PI3Kδ inhibitors. PI3K inhibition had only a minor effect on cell viability and again AKT was reactivated 48h later. Importantly, combining both JAK-kinase inhibition with PI3K inhibition, prevented AKT reactivation and had strong synergistic effect on cell viability and cell proliferation in vitro. Xenotransplantation models of Ph-like ALL with ruxolitinib and INCB-50465 showed a significantly lower leukemia burden as compared to the single agent treatments but disease eradication was not achieved. Pre-BCR signaling can also activate SRC family kinases and we hypothesized that CRLF2-rearanged Ph-like ALL might be sensitive to SRC inhibition as well. Strikingly, combining JAK2, PI3K, and SRC inhibition further reduced the cell viability by one log scale compared to the JAK2/PI3K inhibitor treated samples and less than 0,2% viable cells were detected, while other ALL subtypes were not affected. In vivo validation of treatment strategy is pending. Clinical relevance: While children with ALL currently have a 5-year survival rate of 90%, adults, especially those over the age of 40, have a very poor clinical outcome with survival rates of about 40%. Adults often do not tolerate high doses of chemotherapy regimens and are therefore in clinical need of alternative treatment regimens. We therefore combined JAK1/2 and PI3K inhibitory treatments with dasatinib, a frequently-used tyrosine kinase inhibitor that is well tolerated by older patients. Strikingly, the combinatory treatment had a strong effect on cell viability and represents so far, the first study using specific signaling inhibitors combined in Ph-like ALL. Conclusion: These findings identify that Ph-like ALL are not oncogene addicted like Ph+ ALL. While BCR-ABL1 activates multiple signaling pathways that can be inhibited with ABL kinase inhibitors, Ph-like ALL need to be treated with multiple inhibitors to induce the same effect. Here we show that a combination of JAK, PI3K and SRC-kinase inhibitors is needed to prevent reactivation of signaling pathways and to induce an apoptotic effect that may be clinically relevant. Disclosures Hurtz: Incyte: Research Funding. Tasian: Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Research Funding. Ruggeri: Incyte Corporation: Employment, Equity Ownership. Stubbs: Incyte Corporation: Employment, Equity Ownership. Perl: Daiichi Sankyo: Consultancy; Asana Biosciences: Other: Scientific advisory board; Arog Pharmaceuticals: Consultancy; Novartis: Other: Advisory Board; Seattle Genetics: Other: Advisory board; Astellas: Consultancy; Pfizer: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board. Carroll: Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding.

The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and function by mediating the signaling of key receptors on B cells. Increased malignant B cell proliferation and survival has also been associated with aberrant activation of PI3Kδ, making selective inhibition of this isoform an attractive therapeutic approach for the treatment of B cell malignancies. INCB050465 is a potent inhibitor of PI3Kδ, with a >20,000 fold selectivity over other PI3K isoforms. Emerging clinical data indicate that INCB050465 monotherapy is well tolerated and results in promising clinical responses in patients with various lymphoma histologies, including those with DLBCL. We therefore sought to explore rational combination strategies for INCB050465 using mouse xenograft models of ABC-subtype (HBL-1), GCB-subtype (Pfeiffer), and GCB/double-hit (WILL-2) human DLBCL, evaluating standard of care agents such as bendamustine and rituximab, as well as with targeted agents. PIM inhibition is a logical addition to PI3Kδ inhibition as a therapeutic approach as both kinases play a critical role in the AKT signaling pathway, having overlapping substrates. Likewise BET inhibition is a rational addition to PI3Kδ inhibition in “double-hit” DLBCL due to de-regulation of MYC transcriptional activity. In vivo studies performed in the Pfeiffer xenograft model demonstrate that INCB050465 combined with the pan-PIM inhibitor INCB053914 yielded complete tumor regressions. This profound decrease in tumor cell survival was due in part to the significant reduction in pBAD levels resulting from dual PIM and PI3Kδ inhibition. Despite modest single agent activity in vivo, the combination of INCB050465 with BET inhibitors, INCB054329 or INCB057643, resulted in significant anti-tumor efficacy in all of the DLBCL models studied, and caused a marked repression in tumor MYC expression. To study the transcriptional effects of combining PI3Kδ and BET inhibitors in this lymphoma model, WILL-2 xenograft tumors from mice treated with single dose INCB050465, INCB054329, the combination, or vehicle control were analyzed by RNAseq. INCB050465 enhanced the ability of INCB054329 to repress a MYC-driven transcriptional program, and the combination also regulated multiple developmental and inflammatory pathways. Together, these data support the clinical evaluation of the PI3Kδ inhibitor INCB050465 as part of a combination regimen with PIM or BET inhibitors for the treatment of DLBCL. Citation Format: Matthew C. Stubbs, Robert Collins, Leslie Hall, Alla Volgina, Holly Koblish, Sang Hyun Lee, Timothy Burn, Phillip C. Liu, Jin Lu, Eddy Yue, Yun-Long Li, Andrew P. Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Preclinical studies on potential therapeutic combination partners for the potent and selective PI3Kδ inhibitor INCB050465 in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 143. doi:10.1158/1538-7445.AM2017-143

Inhibitors of the Bromodomain and Extra-Terminal (BET) family of bromodomain containing proteins regulate expression of key cell fate, cell cycle, and survival genes including c-myc. In preclinical models, BET inhibitors have demonstrated significant efficacy in a variety of different oncology indications, including hematological malignancies. Here we describe the preclinical profile of the novel, orally bioavailable BET inhibitor INCB057643 in preclinical models of hematologic malignancies. INCB057643 inhibited binding of BRD2/BRD3/BRD4 to an acetylated histone H4 peptide in the low nM range, and was selective against other bromodomain containing proteins. In vitro analyses showed that INCB057643 inhibited proliferation of human AML, DLBCL, and multiple myeloma cell lines, with a corresponding decrease in MYC protein levels. Cell cycle analyses indicated that G1 arrest and a concentration-dependent increase in apoptosis were seen within 48 hours of treatment with INCB057643. BRD proteins also regulate the expression of many pro-inflammatory genes. Production of several cytokines, including IL-6, IL-10 and MIP-1α, was repressed by INCB057643 in human and mouse whole blood stimulated ex vivo with LPS. Consistent with these effects, analyses of gene expression in cells treated with INCB057643 revealed that pathways involved in cell cycle progression, apoptosis, and IL-6 were among the most significantly altered in vitro. Oral administration of INCB057643 resulted in significant anti-tumor efficacy in xenograft models of AML, myeloma, and DLBCL. Additionally, combining INCB057643 with standard of care agents used for the treatment of DLBCL including rituximab and bendamustine resulted in enhanced anti-tumor efficacy relative to that achieved with single agent therapies at doses that were well tolerated. In addition, many B cell malignancies are reliant on the PI3Kδ pathway for proliferation and survival, suggesting that the combination of INCB057643 with the clinical stage PI3Kδ specific inhibitor INCB050465 may be a rational therapeutic strategy for DLBCL. Compared with single agent BETi or PI3Kδi therapy, the combination significantly potentiated tumor growth inhibition in DLBCL models representative of the ABC subtype (HBL-1), and the double hit GCB subtype (WILL2). These data suggest that clinical exploration of INCB057643 as a monotherapy or in combination in hematologic malignancies is warranted. Citation Format: Matthew C. Stubbs, Thomas Maduskuie, Timothy Burn, Sharon Diamond-Fosbenner, Nikoo Falahatpisheh, Alla Volgina, Nina Zolotarjova, Xiaoming Wen, Patricia Feldman, Mark Rupar, Robert Collins, Cindy Marando, Bruce Ruggeri, Maryanne Covington, Xuesong Mike Liu, Richard Wynn, Swamy Yeleswaram, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Andrew P. Combs, Phillip C. Liu. Preclinical characterization of the potent and selective BET inhibitor INCB057643 in models of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5071. doi:10.1158/1538-7445.AM2017-5071

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with limited treatment options for advanced stage disease. Thus, there is a critical medical need for improved therapies. In approximately 10% of HCC, a focal amplicon at 11q13 harboring FGF19 has been reported. High levels of FGF19 have been shown to drive HCC tumor development and progression in preclinical models, suggesting that selective targeting of FGFR4, a high affinity receptor for FGF19, may be efficacious in these HCC tumors. INCB062079 a potent and selective irreversible inhibitor of FGFR4 (>250-fold vs FGFR1/2/3) suppresses the growth of HCC cell lines driven by amplification and overexpression of FGF19. In subcutaneous xenograft models of HCC, oral dosing of INCB062079 at tolerated doses resulted in dose-dependent inhibition of tumor growth with regressions observed at higher doses consistent with inhibition of FGFR4 signaling in the tumors. In combination with sorafenib, the only approved targeted therapy for HCC, FGFR4 inhibition exhibited additive tumor growth inhibition in the Huh7 model. To assess exposure of INCB062079 in orthotopic tumors after oral dosing, Hep3B tumors were implanted surgically into the liver and their development monitored by analysis of plasma alpha-fetoprotein (AFP). At efficacious doses, INCB062079 strongly suppressed the levels of AFP and FGF19 secreted by the tumors, and their levels correlated well with the reduction in terminal liver tumor mass, suggesting that these may be surrogate markers for response of HCC tumors to INCB062079. In two PDX models of HCC with amplification of FGF19 (4-6 CNV), INCB062079 administration reduced tumor growth by 50-66% at doses that were well tolerated. Additional surrogate markers for FGFR4 inhibition were explored including several parameters related to FGF19 regulation of bile acid metabolism. The mRNA levels of CYP7A1, encoding cholesterol 7a-hydroxlyase, the rate limiting enzyme in bile acid synthesis, were induced in the livers of cynomolgus monkeys upon dosing with INCB062079. Correspondingly there was a dose-dependent increase in fecal bile acids. In summary these data demonstrate that INCB062079 is highly and selectively efficacious in models of HCC with FGF19-FGFR4 oncogene addiction and elicits pharmacodynamic responses in primates providing support for the clinical evaluation of INCB062079 in genetically selected liver cancer patients. Citation Format: Bruce Ruggeri, Matthew Stubbs, Yan-ou Yang, Ashish Juvekar, Liang Lu, Sindy Condon, Darlise DiMatteo, Xiaoming Wen, Paul Collier, Timothy Burn, Liangxing Wu, Daniel Wilson, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Phillip CC Liu. The novel FGFR4-selective inhibitor INCB062079 is efficacious in models of hepatocellular carcinoma harboring FGF19 amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1234. doi:10.1158/1538-7445.AM2017-1234

BACKGROUND Cigarette smoking is among the few unequivocal risk factors for the development of pancreatic ductal adenocarcinoma (PDAC). Activating mutations in codon 12 of the K-ras protooncogene is a frequent and early molecular event in the pathogenesis of PDAC and a variety of nonmalignant ductal pancreatic lesions. The molecular epidemiologic relation between heavy cigarette smoking and mutational activation of K-ras in PDAC has been examined to a limited extent. The authors have examined the mutational status of K-ras in nonneoplastic pancreata in relation to cigarette smoking status. METHODS Archival formalin fixed paraffin embedded specimens of nonneoplastic pancreata (n = 39) were obtained from the American Cancer Society and evaluated histopathologically. Specimens from age- and gender-matched individuals were stratified into three groups: 1) those who never smoked cigarettes (n = 16), 2) those who smoked 1–2 packs/day for more than 20 years (n = 10 cases), and 3) those who smoked more than 2 packs/day for 20 or more years (n = 13). Cases were preselected from 77 specimens based on the quality, suitability, and cellularity of the archival tissues for analyses. Furthermore, none of the patients died of primary PDAC or had evidence of pancreatic metastases from an extrapancreatic primary tumor. Tissue sections were microdissected and deparaffinized, and genomic DNA was purified by standard proteinase K-phenol-chloroform extraction techniques. Genomic DNA was analyzed for mutations in codon 12 of the K-ras protooncogene by two mutant-allele-enriched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays and by multiplex PCR-based ligase chain reaction (LCR) analyses. RESULTS Analyses of multiple microdissected pancreata specimens from 39 cases revealed wild-type K-ras codon 12 sequences in both nonsmoking individuals and those who smoked 1–2 packs/day for 20 or more years. K-ras codon 12 mutations were confirmed by PCR-RFLP and PCR-LCR assays in 5 of 13 pancreata cases (39%) obtained from individuals who smoked more than 2 packs of cigarettes/day for 20 years or more (P < 0.005). The K-ras mutation spectra revealed two GT transversions, one GC transversion and two GA transitions. There was no clear relation between the incidence or spectra of mutations and pancreatic histopathology, as overtly normal pancreata as well as pancreata with squamous metaplasia, periductal fibrosis, and ductal atypia revealed reproducible K-ras alterations. Similarly, among those 34 cases in which a wild-type K-ras sequence was revealed by both approaches, a similar histopathologic profile was evident. CONCLUSIONS Mutational activation of codon 12 of the K-ras protooncogene was confirmed reproducibly by mutant allele-enriched PCR-RFLP and multiplex PCR-LCR analyses in 39% (5 of 13) of archival nonneoplastic pancreata from age- and gender-matched individuals who smoked more than 2 packs of cigarettes/day for 20 or more years. The presence of a mutated or wild-type or K-ras was independent of the histopathologic profile of the 39 cases examined. The data provide further suggestive molecular epidemiologic evidence of an association between a major and unequivocal risk factor for PDAC (heavy cigarette smoking) and mutations in a molecular target (K-ras), the activation of which is an important and early event Cancer 1999;85:326–32. © 1999 American Cancer Society.

Animal models have historically played a critical role in the exploration and characterization of disease pathophysiology, target identification, and in the in vivo evaluation of novel therapeutic agents and treatments. In the wake of numerous clinical trial failures of new chemical entities (NCEs) with promising preclinical profiles, animal models in all therapeutic areas have been increasingly criticized for their limited ability to predict NCE efficacy, safety and toxicity in humans. The present review discusses some of the challenges associated with the evaluation and predictive validation of animal models, as well as methodological flaws in both preclinical and clinical study designs that may contribute to the current translational failure rate. The testing of disease hypotheses and NCEs in multiple disease models necessitates evaluation of pharmacokinetic/pharmacodynamic (PK/PD) relationships and the earlier development of validated disease-associated biomarkers to assess target engagement and NCE efficacy. Additionally, the transparent integration of efficacy and safety data derived from animal models into the hierarchical data sets generated preclinically is essential in order to derive a level of predictive utility consistent with the degree of validation and inherent limitations of current animal models. The predictive value of an animal model is thus only as useful as the context in which it is interpreted. Finally, rather than dismissing animal models as not very useful in the drug discovery process, additional resources, like those successfully used in the preclinical PK assessment used for the selection of lead NCEs, must be focused on improving existing and developing new animal models.

Preclinical models of human cancers are indispensable in the drug discovery and development process for new cancer drugs, small molecules and biologics. They are however imperfect facsimiles of human cancers given the genetic and epigenetic heterogeneity of the latter and the multiplicity of dysregulated survival and growth-regulatory pathways that characterize this spectrum of diseases. This review discusses pre-clinical tumor models - traditional ectopic xenografts, orthotopic xenografts, genetically engineered tumor models, primary human tumorgrafts, and various multi-stage carcinogen-induced tumor models - their advantages, limitations, physiological and pathological relevance. Collectively, these animal models represent a portfolio of test systems that should be utilized at specific stages in the drug discovery process in a pragmatic and hierarchical manner of increasing complexity, physiological relevance, and clinical predictability of the human response. Additionally, evaluating the efficacy of novel therapeutic agents emerging from drug discovery programs in a variety of pre-clinical models can better mimic the heterogeneity of human cancers and also aid in establishing dose levels, dose regimens and drug combinations for use in clinical trials. Nonetheless, despite the sophistication and physiological relevance of these human cancer models (e.g., genetically engineered tumor models and primary human tumografts), the ultimate proof of concept for efficacy and safety of novel oncology therapeutics lies in humans. The judicious interpretation and extrapolation of data derived from these models to humans, and a correspondingly greater emphasis placed on translational medical research in early stage clinical trials, are essential to improve on the current clinical attrition rates for novel oncology therapeutic agents.

Sickle cell disease (SCD) is an autosomal recessive genetic disorder caused by a point mutation in the human β-globin gene. Patients harboring this mutation can exhibit long-chain polymers of hemoglobin and sickle-shaped red blood cells, and suffer from severe medical manifestations including hemolysis and vaso-occlusive crises. Multiple preclinical, clinical and epidemiologic studies have shown that the levels of unmutated fetal hemoglobin (HbF encoded by the γ-globin gene) correlate with less severe disease, validating HbF induction as a therapeutic approach in SCD. Treatment with hydroxyurea (HU), the only approved therapy for SCD, results in a variable induction of HbF and significant improvement in the frequency of pain crises. However, a significant percentage of patients treated with HU fail to exhibit durable benefit, necessitating the need for alternative therapeutic agents. The human γ-globin gene is repressed in the post-natal period by epigenetic mechanisms, and therefore may lend itself to pharmacological intervention aimed at derepressing gene expression. One of the most important of these epigenetic mechanisms is catalyzed by lysine-specific demethylase 1 (LSD1), a histone demethylase that removes mono-/dimethyl marks from the lysine 4 and 9 residues of histone H3 through an FAD-directed redox process. Here, we report the characterization of selective, potent, and orally bioavailable LSD1 inhibitors from two classes - FAD-directed inhibitors that achieve inhibitory activity through formation of covalent FAD-adducts and non-FAD-directed, reversible inhibitors - and demonstrate their ability to induce γ-globin gene expression in murine and primate preclinical models. In the Towne's SCD mouse model, oral administration of LSD1 inhibitors significantly increased HbF+ cell (F cell) production. Concurrent with the increase in F cells, sickle cell numbers, reticulocyte counts, and bilirubin levels were all markedly reduced, indicating an amelioration of several pathophysiological features of SCD. FAD- and non-FAD-directed LSD1 inhibitors were more effective than HU in increasing F cells production, and the combination of HU and suboptimal doses of LSD1 inhibitors resulted in a greater induction of F cells and more pronounced reductions in reticulocyte counts and bilirubin levels. In addition to the humanized SCD model, HbF induction in response to LSD1 inhibitor treatment was evaluated in non-anemic cynomolgus monkeys. Oral administration of LSD1 inhibitors significantly induced F cells and HbF in a dose-dependent manner and over a sustained period (>50 days) following the discontinuation of treatment. The percentage of induced F cells in total RBCs was linearly correlated with the percentage of HbF protein induced by LSD1 inhibition. Taken together, these results support the potential utility of LSD1 inhibition as a novel therapeutic approach to increase HbF production. Disclosures Lee: Incyte Corporation: Employment, Other: Stock. Soloviev:Incyte Corporation: Employment, Other: Stock. Zhang:Incyte Corporation: Employment, Other: Stock. Roman:Incyte Corporation: Employment, Other: Stock. Yang:Incyte Corporation: Employment, Other: Stock. Bowman:Incyte Corporation: Employment, Other: Stock. Burke:Incyte Corporation: Employment, Other: Stock. Margulis:Incyte Corporation: Employment, Other: Stock. O'Connor:Incyte Corporation: Employment, Other: Stock. Yang:Incyte Corporation: Employment, Other: Stock. Wu:Incyte Corporation: Employment, Other: Stock. Wynn:Incyte Corporation: Employment, Other: Stock. Burn:Incyte Corporation: Employment, Other: Stock. Shuey:Incyte Corporation: Employment, Other: stock. Diamond:Incyte Corporation: Employment, Other: Stock. Yao:Incyte Corporation: Employment, Other: Stock. Hollis:Incyte Corporation: Employment, Other: Stock. Yeleswaram:Incyte Corporation: Employment, Other: Stocks. Roberts:Incyte Corporation: Employment, Other: Stock. Huber:Incyte Corporation: Employment, Other: Stock. Scherle:Incyte Corporation: Employment, Other: Stock. Ruggeri:Incyte Corporation: Employment, Other: Stock.

Methylated histone marks on H3K4 and H3K9 are generally coupled with transcriptional activation and repression, respectively. Altered levels of these histone methylation marks lead to abnormal gene expression and are associated with oncogenesis. Lysine specific demethylase 1 (LSD1) catalyzes demethylation of mono and di-methylated lysine 4 and 9 of histone H3 via an FAD-dependent redox-process. Deregulated LSD1 activity perturbs normal gene expression and can lead to cellular transformation. In particular, the function of LSD1 has been reported to maintain stem cell-like gene expression patterns in various cancers, most notably small cell lung cancer (SCLC), a cancer type that is characterized by a highly undifferentiated state and with high unmet medical need. INCB059872 is a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. The effect of INCB059872 in SCLC cell lines was assessed in vitro and in vivo. In these studies, the proliferation of a panel of SCLC cell lines was inhibited by INCB059872, with EC50 values ranging from 47 to 377 nM. Non-tumorigenic cells, such as IL-2 stimulated T cells from normal donors, by contrast, were significantly less sensitive with IC50 values > 10 μM. Oral administration of INCB059872 on once daily (QD) and alternative day (QoD) dosing regimens inhibited tumor growth in the NCI-H526 and NCI-H1417 human SCLC xenograft models. Consistent with previous reports, INCB059872 treatment in these models led to the induction of the FEZ1 and UMODL1 genes relative to vehicle-treated animals, as part of a gene signature in SCLC cell lines that is predictive of LSD1 responsiveness. Moreover, serum levels of the neuroendocrine marker pro-GRP were markedly reduced at all efficacious dosing regimens in the NCI-H1417 human SCLC xenograft model, suggesting that serum pro-GRP levels could be used as a surrogate pharmacodynamic marker of LSD1 inhibition. Currently, the efficacy of INCB059872 in combination with standard of care therapies for SCLC is under evaluation in these preclinical models. These data support the clinical evaluation of INCB059872 in patients with SCLC. Citation Format: Sang Hyun Lee, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Margaret Favata, Chunhong He, Liangxing Wu, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. The evaluation of INCB059872, an FAD-directed inhibitor of LSD1, in preclinical models of human small cell lung cancer. [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 4704.

Inhibitors of the BET family of Bromodomain proteins have been shown to be growth inhibitory across a spectrum of tumor types due to their ability to regulate expression of key survival and cell fate determining genes such as c-myc. Among the various tumor histologies, hematologic malignancies are among the most sensitive cancers to BET inhibition. INCB054329 is a novel, non-benzodiazepine, selective BET inhibitor that is undergoing Phase 1 clinical trials and that has shown encouraging in vitro and in vivo preclinical activity in several models of hematologic malignancy. In the current study, the activity of INCB054329 was evaluated in models of B cell malignancy. INCB54329 effectively inhibited the in vitro growth of a panel of cell lines representing both Hodgkin and non-Hodgkin lymphoma. Treated cells arrested primarily in G1 with sensitive lines also exhibiting dose and time-dependent apoptosis. Within a panel of double-hit lymphoma cell lines, which have activating chromosomal rearrangements in both c-myc and bcl-2, INCB054329 potently inhibited cell growth and was more effective than antagonists of BTK, bcl-2, PIM and PI3Kδ. INCB054329 also showed in vivo efficacy in models of diffuse large B-cell lymphoma (DLBCL). As a single agent, oral administration of INCB054329 inhibited tumor growth in Pfeiffer (GBC) and WILL-2 (GCB, double-hit) subcutaneous xenograft models. The in vivo combination of bendamustine with INCB054329 enhanced anti-tumor efficacy compared with either agent alone in the Pfeiffer model, and the combination was well tolerated. A rational, targeted combination strategy was evaluated involving INCB054329 and a selective, orally active PI3Kδ inhibitor, INCB050465, which is currently in clinical trials in B cell malignancies. Combining INCB054329 with PI3Kδ inhibition markedly enhanced anti-tumor efficacy, increasing the incidence of partial tumor regressions in vivo. In this model, both INCB054329 and INCB050465 treatment led to a reduction in c-Myc protein levels, suggesting a convergence between modulation of BET transcriptional regulation and the PI3Kδ pathway. These data suggest that clinical investigation of INCB054329, both as monotherapy and in combination with standard of care or novel targeted therapies, in several classes of B cell lymphoma, including high risk double hit lymphoma, is warranted. Citation Format: Matthew Stubbs, Robert Collins, Alla Volgina, Mike Liu, Margaret Favata, Mark Rupar, Xiaomng Wen, Richard Sparks, Thomas Maduskuie, Maryanne Covington, Timothy Burn, Bruce Ruggeri, Andrew P. Combs, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip CC Liu. Activity of the BET inhibitor INCB054329 in models of lymphoma. [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 3780.

Preclinical and clinical studies have shown that proteasome inhibitors (PIs) have anti-MM activity in combination with dexamethasone or lenalidomide. However, no data exists on the anti-MM effects of combinations involving the PI delanzomib with dexamethasone and/or lenalidomide. Herein, we show that delanzomib in combination with dexamethasone and/or lenalidomide results in superior tumor reduction and extended tumor growth delays when compared to vehicle alone, these drugs alone, or the doublet of dexamethasone and lenalidomide. The favorable results obtained from the three xenograft studies suggest that delanzomib in combination with dexamethasone and lenalidomide should be explored for the treatment of MM.

Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60% to 70% of melanomas, 29% to 83% of papillary thyroid carcinomas, 4% to 16% colorectal cancers, and a lesser extent in serous ovarian and non–small cell lung cancers. The V600E mutation is found in the vast majority of cases and is an activating mutation, conferring transforming and immortalization potential to cells. CEP-32496 is a potent BRAF inhibitor in an in vitro binding assay for mutated BRAFV600E (Kd BRAFV600E = 14 nmol/L) and in a mitogen-activated protein (MAP)/extracellular signal–regulated (ER) kinase (MEK) phosphorylation (pMEK) inhibition assay in human melanoma (A375) and colorectal cancer (Colo-205) cell lines (IC50 = 78 and 60 nmol/L). In vitro, CEP-32496 has multikinase binding activity at other cancer targets of interest; however, it exhibits selective cellular cytotoxicity for BRAFV600E versus wild-type cells. CEP-32496 is orally bioavailable in multiple preclinical species (>95% in rats, dogs, and monkeys) and has single oral dose pharmacodynamic inhibition (10–55 mg/kg) of both pMEK and pERK in BRAFV600E colon carcinoma xenografts in nude mice. Sustained tumor stasis and regressions are observed with oral administration (30–100 mg/kg twice daily) against BRAFV600E melanoma and colon carcinoma xenografts, with no adverse effects. Little or no epithelial hyperplasia was observed in rodents and primates with prolonged oral administration and sustained exposure. CEP-32496 benchmarks favorably with respect to other kinase inhibitors, including RAF-265 (phase I), sorafenib, (approved), and vemurafenib (PLX4032/RG7204, approved). CEP-32496 represents a novel and pharmacologically active BRAF inhibitor with a favorable side effect profile currently in clinical development. Mol Cancer Ther; 11(4); 930–41. ©2012 AACR.

Accumulating evidence suggests that autoreactive plasma cells play an important role in systemic lupus erythematosus (SLE). In addition, several proinflammatory cytokines promote autoreactive B cell maturation and autoantibody production. Hence, therapeutic targeting of such cytokine pathways using a selective JAK2 inhibitor, CEP-33779 (JAK2 enzyme IC50 = 1.3 nM; JAK3 enzyme IC50/JAK2 enzyme IC50 = 65-fold), was tested in two mouse models of SLE. Age-matched, MRL/lpr or BWF1 mice with established SLE or lupus nephritis, respectively, were treated orally with CEP-33779 at 30 mg/kg (MRL/lpr), 55 mg/kg or 100 mg/kg (MRL/lpr and BWF1). Studies included reference standard, dexamethasone (1.5 mg/kg; MRL/lpr), and cyclophosphamide (50 mg/kg; MRL/lpr and BWF1). Treatment with CEP-33779 extended survival and reduced splenomegaly/lymphomegaly. Several serum cytokines were significantly decreased upon treatment including IL-12, IL-17A, IFN-α, IL-1β, and TNF-α. Anti-nuclear Abs and frequencies of autoantigen-specific, Ab-secreting cells declined upon CEP-33779 treatment. Increased serum complement levels were associated with reduced renal JAK2 activity, histopathology, and spleen CD138+ plasma cells. The selective JAK2 inhibitor CEP-33779 was able to mitigate several immune parameters associated with SLE advancement, including the protection and treatment of mice with lupus nephritis. These data support the possibility of using potent, orally active, small-molecule inhibitors of JAK2 to treat the debilitative disease SLE.

A series of novel 7-amino-1,3,4,5-tetrahydrobenzo[b]azepin-2-one derivatives within the diaminopyrimidine class of kinase inhibitors were identified that target anaplastic lymphoma kinase (ALK). These inhibitors are potent against ALK in an isolated enzyme assay and inhibit autophosphorylation of the oncogenic fusion protein NPM-ALK in anaplastic large cell lymphoma (ALCL) cell lines. The lead inhibitor 15, which incorporates a bicyclo[2.2.1]hept-5-ene ring system in place of an aryl moiety, activates the pro-apoptotic caspases (3 and 7) and displays selective cytotoxicity against ALK-positive ALCL cells. Furthermore, 15 provides more than 40-fold selectivity against the structurally related insulin receptor, is orally bioavailable in multiple species, and displays in vivo antitumor efficacy when dosed orally in ALK-positive ALCL tumor xenografts in Scid mice.

Colorectal cancer is the second leading cause of cancer deaths in the United States with an estimated 150,000 diagnosed cases and over 56,000 fatalities annually (Jemal et al., 2006). Approximately one-third to one-half of cases are localized to the colon and rectum and have a favorable prognosis, while one-third to one-half present with regional lymph node metastases at diagnosis and generally are refractory to various chemotherapeutic regimens. Treatment options (surgery, radiation, and chemotherapy) are limited and the disease carries a grave prognosis for many patients. An orthotopic model of colon carcinoma in mice provides a way to evaluate the pathogenesis of tumor growth and metastasis as an aid in developing effective therapies and to better understand the underlying biology of colon tumor growth and metastasis. The protocol described in this unit details the development and characterization of an orthotopic model of murine colon carcinoma in BALB/c mice with diffuse lymphatic and hepatic metastatic spread, closely mimicking the course of the human disease. Curr. Protoc. Pharmacol. 38:14.5.1-14.5.13. © 2007 by John Wiley & Sons, Inc. Keywords: colon carcinoma; athymic nude; BALB/c mice; orthotopic; metastasis

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related deaths in the United States, with a 5-year survival rate of only 2% to 10%. This tumor is aggressive, often metastasizing to distant sites (liver, lung, and adjacent intestines) by the time of diagnosis. Treatment options are limited, and the disease carries a grave prognosis for most patients. An orthotopic model of human PDAC in nude mice provides an excellent way to evaluate the pathogenesis of tumor growth and metastasis in order to develop therapies, to better define the underlying biology of tumor growth and metastasis, and to identify new molecular targets. This unit describes an orthotopic model of human PDAC in athymic nude mice that closely mimics the human condition. It is characterized by diffuse peritoneal, lymphatic, and hepatic metastatic spread and manifestations of a cancer cachexic phenotype. Keywords: Human pancreatic ductal adenocarcinoma; Athymic nude mice; Orthotopic; Metastasis; cachexia

Inflammatory breast cancer (IBC) is a very aggressive type of advanced breast cancer with a poor prognosis. Due to its propensity to rapidly metastasize, IBC is responsible for a disproportionate number of breast cancer-related deaths; 15% of all breast cancer deaths result from this disease. Approximately 30% of IBC patients have distant metastases at time of diagnosis, in contrast to 5% of patients with non-IBC breast cancers. Currently, there is no adequate adjuvant therapy to reduce the risk of recurrence and mortality in IBC patients. We recently found that FAK1, also known as PTK2, is amplified, up-regulated and phosphorylated (active) in some inflammatory breast cancer cells [1]. The focal adhesion kinase FAK1 is a cytoplasmic tyrosine kinase that localizes to focal adhesions, and controls a number of cell pathways including proliferation, viability and survival. Activated FAK1 is absent or low in normal tissue and benign neoplasms and up-regulated in invasive and metastatic tumors. In this study, we investigated a new dual FAK1/ALK inhibitor, CEP-37440, in vitro and in vivo using preclinical models of IBC. We found that 300 nM CEP-37440 was able to decrease the proliferation of the triple negative FC-IBC02 cell line and, 1000 nM CEP-37440 was able to complete inhibit the proliferation of these cells. SUM190 showed a 50% decreased in proliferation when these cells were treated with 1000 nM CEP-37440. Genes involved in functions such as cellular growth and proliferation, cell cycle, cell death and survival and cellular movement and cell-to cell signaling and interaction were differentially regulated by CEP-37440 in sensitive IBC cell lines. Among them, TGFβ1 and MMP3 were down-regulated and DKK3, CAV1 and TFPI2 were up-regulated by CEP-37440. In FC-IBC02 orthotopic breast cancer xenograft models, CEP-37440 was able to reduce the growth of the primary tumor and inhibit the development of spontaneous metastases in brain. In conclusion, CEP-37440 was highly effective in reducing the proliferation in vitro and in vivo of IBC cells that expressed high levels of phospho-FAK (Tyr397). References 1. Fernandez S, Robertson F, Pei J, Aburto-Chumpitaz L, Mu Z, Chu K, Alpaugh K, Huang Y, Cao Y, Ye Z et al: Inflammatory breast cancer (IBC): clues for targeted therapies. Breast Cancer Res Treat 2013, 140:23-33. Citation Format: Israa Salem, Lucy Aburto, Sankar Addya, Inna Chervoneva, Zhaomei Mu, Bruce Ruggeri, Massimo Cristofanilli, Sandra V. Fernandez. CEP-37440, a highly selective and potent dual inhibitor of ALK and FAK1 inhibits the proliferation of inflammatory breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3232. doi:10.1158/1538-7445.AM2015-3232

CEP-7055, a fully synthetic, orally active N,N-dimethylglycine ester of CEP-5214, a C3-(isopropylmethoxy)–fused pyrrolocarbazole with potent pan–vascular endothelial growth factor receptor (VEGFR) kinase inhibitory activity, has recently completed phase I clinical trials in cancer patients. These studies evaluated the antitumor efficacy of CEP-7055 using orthotopic models of glioblastoma and colon carcinoma in combination with temozolomide, and irinotecan and oxaliplatin, respectively, for their effects on primary and metastatic tumor burden and median survival. Chronic administration of CEP-7055 (23.8 mg/kg/dose) and temozolomide resulted in improvement of median survival of nude mice bearing orthotopic human glioblastoma xenografts compared with temozolomide alone (261 versus 192 days, respectively; P ≤ 0.02). Reductions in neurologic dysfunction, brain edema, hemorrhage, and intratumoral microvessel density (CD34 staining) were observed in glioma-bearing mice receiving CEP-7055 alone, temozolomide alone, and the combination of CEP-7055 and temozolomide relative to vehicle and to temozolomide monotherapy. The administration of CEP-7055 in combination with irinotecan (20 mg/kg/dose i.p. × 5 days), and to a lesser degree with oxaliplatin (10 mg/kg/dose i.v.), showed reductions on primary colon carcinoma and hepatic metastatic burden in the CT-26 tumor model relative to that achieved by irinotecan and oxaliplatin monotherapy. These data show the significant efficacy and tolerability of optimal efficacious doses of CEP-7055 when given in combination with temozolomide and irinotecan relative to monotherapy with these cytotoxic agents in preclinical orthotopic glioma and colon carcinoma models and lend support for the use of these treatment regimens in a clinical setting in patients with glioblastoma and colon carcinoma. [Mol Cancer Ther 2006;5(7):1744–53]

The survival rate for patients with pancreatic ductal adenocarcinoma (PDAC) is among the poorest for all cancers. The factors that contribute to this poor prognosis are lack of effective early detection, high rate of metastases and a generally refractory response to available treatment modalities. The most commonly used treatment methods--chemotherapy and radiation therapy--are mainly used for symptom palliation, with surgery being the only "curative" treatment option. The use of combinations of treatment modalities is the only therapy available to patients with locally advanced disease or that which is surgically unresectable. These options are still not sufficient to increase patient survival time significantly. The aggressive behavior and poor prognosis of this cancer is associated with an increased expression of many growth factors and their cognate receptors. We have demonstrated previously the aberrant expression of the Trk receptors (Trks A, B, and C) in PDAC specimens and human PDAC-derived cell lines and a biphasic, dose-dependent response of specific neurotrophic agents on the in vitro invasiveness of PDAC cells. Based on these data we have evaluated the therapeutic potential of inhibiting neurotrophin-Trk interactions using a selective Trk tyrosine kinase inhibitor (CEP-701) on subcutaneous (s.c.) and tracheal xenografts derived from the poorly differentiated PDAC cell line, Panc1. We demonstrate that CEP-701 administration at 10 mg/kg s.c. BID for 21 days inhibited tumor growth of the Panc1 s.c. xenografts in a statistically-significant manner (p < 0.01) compared to vehicle controls, in the absence of morbidity and mortality. A T/C value of 25% was observed for CEP-701-treated s.c. xenografts. In addition, CEP-701 administration inhibited tumor cell invasion in the s.c. tracheal xenograft model of in vivo invasiveness. Taken together, these data suggest that further studies are warranted to evaluate CEP-701 as a potential therapeutic agent in the treatment of PDAC.

The oncogenic receptor tyrosine kinases (RTK) AXL and c-Met are over-expressed and constitutively activated in a variety of human cancers. Activation of these receptors contributes to multiple steps in tumor progression, including the epithelial-mesenchymal transition (EMT) by which cancer cells escape to form metastases and frequently develop resistance to a variety of agents, most notably to epidermal growth factor receptor (EGFR) targeted therapies. CEP-40783 is an orally-active, nanomolar potent and highly kinase-selective Type II inhibitor of the AXL and c-Met RTK in drug development. In the current study, five Champions human TumorGraft™ models of NSCLC expressing constitutively activated AXL and/or c-Met were used to determine the anti-tumor efficacy of single agent CEP-40783 compared to paclitaxel and the EGFR inhibitor, erlotinib (Genentech-Roche; OSI Pharmaceuticals). Champions Oncology has developed an innovative platform that utilizes the implantation of primary human tumors in immune-deficient mice in a manner that preserves the biological properties of the original human tumor and results in more rigorous and clinically relevant models for drug discovery. Previous reports have shown the correlation between responsiveness observed in Champions TumorGraft™ models and clinical responses of the patients from which the models were derived. Mice bearing established Champions TumorGrafts™ were treated orally with 10 mg/kg and 30 mg/kg qd of CEP-40783 for 10 to 34 days (until control tumors achieved >1000 mm3 volume) and anti-tumor efficacy and tolerability were evaluated. In 3/5 (60%) of the tumor models, CEP-40783 showed in vivo efficacy, including tumor regressions, significantly superior to that achieved with an optimal regimen of paclitaxel. In 4/4 (100%) of the erlotinib-insensitive tumor models, CEP-40783 demonstrated significant efficacy (66 to 118% TGI) compared to the control group at the 30 mg/kg dose. Additionally, CEP-40783 in combination with erlotinib demonstrated superior anti-tumor efficacy compared to CEP-40783 and erlotinib single agents in the one erlotinib-sensitive model evaluated. CEP-40783 as a single agent and in combination with erlotinib was well tolerated. Retrospective bioinformatics analyses are planned to determine potential signatures of response and resistance to CEP-40783. These results demonstrate that CEP-40783 shows superior efficacy alone and in combination with erlotinib in Champions TumorGraft™ models of NSCLC compared to SOC agents and supports the development of CEP-40783 for the treatment of erlotinib-resistant NSCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C272. Citation Format: Jay A. Friedman, Rodney Donaldson, Sheila Miknyoczki, Mangeng Cheng, Robert Hudkins, Bruce Dorsey, Mark Ator, Thelma Angeles, Bruce Ruggeri, Elizabeth Bruckheimer. Antitumor activity of the dual AXL/c-Met inhibitor CEP-40783 in Champions primary TumorGraft™ models of human non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C272.

Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma that includes two entities ALK+ and ALK- ALCL based on chromosomal translocations of the anaplastic lymphoma kinase (ALK). ALK- ALCL have very poor clinical outcome with conventional chemotherapy. Lack of representative cell lines and/or models has compromised the development of successful therapy. Toward this end, we report the generation and characterization of a group of novel ALCL tumor grafts. We implanted subcutaneously (s.c.) tumor tissue fragments, fresh or viable cryopreserved, from 11 cases of primary systemic ALCL (3 ALK+ and 8 ALK-) in six- to eight-week old severe immunocombined immunodeficiency NOD Cg-Prkdcscid/Il2rgtm1wjl/SzJ (NSG) mice. A single leukaemic ALCL sample was injected i.v. or s.c. Six cases led to successful lymphoma growth within 6-12 weeks as subcutaneous solid spheroid masses constituted by a uniform population of large pleomorphic cells CD30-positive. All tumor grafts were re-implanted in recipient animals, in same cases up to 21 consecutive implants with an analogous time growth rate. Two cases, in the same way of the original human neoplasia, were early associated with concomitant enlargement of multiple organs. All tumor graft lines eventually showed disseminate disease with involvement of distant lymph nodes and of spleen (with homig in peri-arteriolar spaces), liver (initially observed within the sinusoidal spaces) and kidney. The morphological and immunohistochemical (IHC) evaluation (expression of CD30, cytotoxic proteins and ALK) of the serially propagated ALCL tumor grafts demonstrated a high concordance between primary and derived animal tumors which showed highly stable profile along different passages from the early generation (T1) until the final ones (up to T16). The clonal relationship with primary and corresponding tumor grafts was confirmed by gene TCR rearrangement analyses and by ALK FISH studies. In addition we interrogated the model using gene expression profiling and single nucleotide polymorphism which established a close relationship among primary and derived tumors. Massive parallel sequencing on mRNA transcripts defines the presence of two novel chimeras involving NF1-MAZ and TRAF1-ALK1, which confirmes the evidence of ALK signaling and pinpoint a role of RAS in ALK- ALCL. The tumor grafts displayed, also, therapeutic responses with conventional therapy (CHOP) which reflected that seen in the donor patients: initial partial responses followed invariably by clinical relapses. To overcome this refractory we assayed the sensitivity of two tumorgraft lines, generated from ALK+ ALCL, to a new selective ALK inhibitor (CEP28122) that led a rapid and stable response. This study demonstrated the key role of these libraries of tumor grafts, especially in developing and testing new therapeutic regimens in refractory ALCL patients. Citation Format: Fabrizio Tabbo’, Antonella Barreca, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Francesco Abate, Michela Boi, Maria Todaro, Cristina Abele, Domenico Novero, Alberto Zamo’, Marco Chilosi, Maurilio Ponzoni, Mangeng Cheng, Bruce Ruggeri, Pierpaolo Piccaluga, Stefano Pileri, Enrico Tiacci, Brunangelo Falini, Lenny Shultz, Roberto Piva, Enzo Medico, Francesco Bertoni, Raul Rabadan, Giorgio Inghirami. Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3853. doi:10.1158/1538-7445.AM2013-3853

Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60-70% of melanomas, 23-83% of papillary thyroid carcinomas, 4-16% colorectal cancers, and a lesser extent in serous ovarian and non-small-cell lung cancers. The V600E mutation is found in the vast majority of these cases and is an activating mutation, conferring transforming and immortalization potential to cells. CEP-32496 is a potent BRAF inhibitor that demonstrates selective anti-tumor activity against BRAFV600E mutated cancer cells both in vitro and in vivo. CEP-32496 is orally bioavailable and demonstrates in vivo pharmacodynamic inhibition of pMEK and pERK signaling and tumor stasis and regressions in BRaf V600E cell line tumor xenograft models. In the current study Champions primary human TumorGraft models of melanoma and colorectal carcinoma possessing the B-Raf V600E genotype were utilized to determine the oral anti-tumor efficacy of CEP-32496 in more clinically relevant models in order to support the clinical development of CEP-32496. Champions Oncology has developed an innovative platform for oncology research that utilizes the implantation of primary human tumors in immune-deficient mice in a manner that preserves the biological properties of the original human tumor. Previous reports have demonstrated the correlation between the responses observed in Champions TumorGraft™ models and clinical responses from the patients from which the models were derived. CEP-32496 was evaluated in a panel of nine Champions TumorGraft models and compared to the recently FDA-approved B-RAF inhibitor, vemurafenib (Roche/Genentech/Daiichi) in order to determine the potential clinical activity of the compound. Mice bearing established Champions TumorGrafts were treated orally with 35 mg/kg bid of CEP-32496 or vemurafenib for 25 to 35 days and anti-tumor efficacy and tolerability were evaluated. Results demonstrated that in 5/9 (56% of the Tumorgraft models) CEP-32496 showed significantly superior in vivo efficacy compared to vemurafenib. Equivalent anti-tumor efficacy was observed in 3/9 (33%) TumorGraft models with both CEP-32496 and vemurafenib. Both inhibitors demonstrated comparable tolerability profiles. Additional TumorGraft models are currently under evaluation and retrospective bioinformatics analyses are planned to determine potential signatures of response and resistance for CEP-32496 to facilitate both its clinical development and potential differentiation from vemurafenib. Overall, these results demonstrate that CEP-32496 shows comparable to superior efficacy in this panel of Champions primary TumorGraft models when compared to vemurafenib. 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 3755. doi:1538-7445.AM2012-3755

Anaplastic lymphoma kinase (ALK) was originally identified as the oncogenic NPM (nucleophosmin)-ALK fusion protein due to a t (2;5) chromosomal translocation in anaplastic large cell lymphomas (ALCL). Many other chromosomal rearrangements or gene mutations/amplification leading to enhanced ALK activity have subsequently been identified and characterized in a number of human cancer types. The recent reports of EML4 (echinoderm microtubule-associated protein-like 4)-ALK oncogenic proteins in non-small cell lung cancer (NSCLC) and the identification of ALK activating point mutations and gene amplification in neuroblastoma have indicated ALK as a potential major therapeutic target for human cancers. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity (IC50 of 3 nM) and NPM-ALK tyrosine phosphorylation in ALCL cells (IC50 of 20-30 nM). It is selective over a broad panel of protein kinases and a panel of receptors and ion channels with greater than 300-fold selectivity for insulin receptor. CEP-28122 induced concentration-dependent growth inhibition and cytotoxicity of ALK-positive ALCL and NSCLC cells with minimal activity against ALK-negative lymphoma and leukemia cells as well as ALK-negative NSCLC cells at concentrations up to 3 μM. CEP-28122 exhibited favorable oral bioavailability (F = 37-71% across species) with adequate tissue distribution in rodents. It displayed dose-dependent inhibition of NPM-ALK tyrosine phosphorylation in human ALCL tumor xenografts in mice with complete target inhibition (> 90%) for more than 12 h following single oral dosing at 30 mg/kg. Dose-dependent anti-tumor activity was observed in NPM-ALK-positive Sup-M2 and Karpas-299 ALCL tumor xenografts and EML4-ALK-positive NCI-H2228 and NCI-H3122 tumor xenografts in mice dosed with CEP-28122, bid, po, with complete or near complete tumor regressions observed following 2 weeks of treatment with CEP-28122 at 30 mg/kg or higher. Treatment of mice bearing Sup-M2 tumor xenografts or primary human ALCL tumorgrafts with CEP-28122 at 55 or 100 mg/kg bid, po, for 4 weeks led to sustained tumor regression in all mice, with no tumor re-emergence in any mouse up to 60 days post cessation of CEP-28122 treatment. On the contrary, CEP-28122 displayed marginal anti-tumor activity against ALK-negative lymphoma and NSCLC tumor xenografts under the same dosing regimens. It was well tolerated with all dosing regimens in mice and rats with no overt toxicity and no compound-related body weight loss. CEP-28122 advanced into preclinical development based on its potency, selectivity and overall favorable pharmacological, pharmaceutical and safety profiles. 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 3574. doi:10.1158/1538-7445.AM2011-3574

Background Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis. We recently found that focal adhesion kinase 1 (FAK1) is upregulated and phosphorylated (active) in IBC. In this study, we investigated the effect of CEP-37440, a dual inhibitor of FAK1 and anaplastic lymphoma kinase (ALK), using human IBC cell lines and preclinical models of IBC. Methods Cell proliferation assays were performed in the presence of several concentrations of CEP-37440 using IBC and triple-negative breast cancer non-IBC cell lines. In vitro, we studied the expression of total FAK1, phospho-FAK1 (Tyr 397), total ALK and phospho-ALK (Tyr 1604). In vivo, we tested CEP-37440 using FC-IBC02, SUM149, and SUM190 IBC xenograft mouse models. Results CEP-37440 at low concentration decreased the proliferation of the IBC cell lines FC-IBC02, SUM190, and KPL4, while not affecting the proliferation of normal breast epithelial cells. At higher concentration, CEP-37440 was also able to inhibit the proliferation of the IBC cell line MDA-IBC03 and the triple-negative non-IBC cell lines MDA-MB-231 and MDA-MB-468; the IBC cell line SUM149 showed a slight response to the drug. CEP-37440 decreased the cell proliferation of FC-IBC02, SUM190, and KPL4 by blocking the autophosphorylation kinase activity of FAK1 (Tyr 397). None of the cells evaluated expressed ALK. In vivo, after 7 weeks of CEP-37440 treatment, the SUM190, FC-IBC02, and SUM149 breast tumor xenografts were smaller in mice treated with 55 mg/kg bid CEP-37440 compared to the controls; the tumor growth inhibition (TGI) was 79.7 %, 33 %, and 23 %, respectively. None of the FC-IBC02 breast xenografts mice treated with CEP-37440 developed brain metastasis while 20 % of the mice in the control group developed brain metastasis. Expression array analyses in FC-IBC02 cells showed that CEP-37440 affects the expression of genes related to apoptosis, interferon signaling, and cytokines. Conclusions CEP-37440 is effective against some IBC cells that express phospho-FAK1 (Tyr 397), and its antiproliferative activity is related to its ability to decrease phospho-FAK1. Our results suggest that combinational therapies could be more effective than using CEP-37440 as a single agent. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0694-4) contains supplementary material, which is available to authorized users.

Introduction Janus kinase 2 (JAK2) is involved in the downstream activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 and is responsible for transducing signals for several proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), including interleukin (IL)-6, interferon γ (IFNγ) and IL-12. In this paper, we describe the efficacy profile of CEP-33779, a highly selective, orally active, small-molecule inhibitor of JAK2 evaluated in two mouse models of RA. Methods Collagen antibody-induced arthritis (CAIA) and collagen type II (CII)-induced arthritis (CIA) were established before the oral administration of a small-molecule JAK2 inhibitor, CEP-33779, twice daily at 10 mg/kg, 30 mg/kg, 55 mg/kg or 100 mg/kg over a period of 4 to 8 weeks. Results Pharmacodynamic inhibition of JAK2 reduced mean paw edema and clinical scores in both CIA and CAIA models of arthritis. Reduction in paw cytokines (IL-12, IFNγ and tumor necrosis factor α) and serum cytokines (IL-12 and IL-2) correlated with reduced spleen CII-specific T helper 1 cell frequencies as measured by ex vivo IFNγ enzyme-linked immunosorbent spot assay. Both models demonstrated histological evidence of disease amelioration upon treatment (for example, reduced matrix erosion, subchondral osteolysis, pannus formation and synovial inflammation) and reduced paw phosphorylated STAT3 levels. No changes in body weight or serum anti-CII autoantibody titers were observed in either RA model. Conclusions This study demonstrates the utility of using a potent and highly selective, orally bioavailable JAK2 inhibitor for the treatment of RA. Using a selective inhibitor of JAK2 rather than pan-JAK inhibitors avoids the potential complication of immunosuppression while targeting critical signaling pathways involved in autoimmune disease progression.