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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.

Adenoid cystic carcinoma (ACC) is a rare cancer of secretory glands, characterized by slow and unpredictable growth, unrelenting relapse and high rates of metastasis. Poor response to chemotherapy and targeted drugs has resulted in there being no approved therapies to date, which contributes to poor prognosis with less than a 20% 5-year survival rate in patients with high-risk ACC. A vast majority of ACC tumors are known to have recurrent chromosomal translocation t(6;9) or t(8;9) resulting in MYB/MYBL1-NFIB fusions, and high levels of MYB protein expression. Furthermore, a subset of ACC tumors expresses NOTCH1 activating mutations (20-25%) and hyperactive NOTCH signaling in both primary and recurrent/metastatic tumors. A previous report has demonstrated that an inhibitor of PRMT5 (protein arginine methyltransferase) shows favorable responsiveness in patients with advanced ACC in a phase I clinical trial. Here, we investigate mechanistic aspects as to how PRMT5 inhibition potentially regulates the unique molecular drivers of ACC tumor growth. Due to a limited number of validated in vitro cellular models, we selected a group of head and neck cancer cell lines, including those originating from salivary gland, along with MYB and NOTCH1-driven leukemia models, to evaluate the effects of PRMT5 inhibition on MYB and NOTCH regulated genes. Treatment with PRT543, a potent and selective PRMT5 inhibitor, decreased expression of MYB and its associated downstream gene GATA3, as well as ACC-related genes such as SOX4 and POU3F2. Furthermore, PRT543 downregulated MYB signaling (MYB, FOXM1, NIK and SKI) as well as MYB alternative isoforms MYB-9A and MYB-10A in MYB expressing leukemia cells. Intriguingly, treatment with PRT543 also downregulated the transcription factor, HSF4, that is uniquely linked to regulation by N-terminal truncated MYB, a variant driven by an alternative promoter (TSS2) and reported as being active in ACC tumors. In addition to regulation of MYB, we also show that PRT543 downregulates NOTCH1 expression as well as multiple NOTCH1 target genes in leukemia cells which harbor a NOTCH1 active mutation. Given these results, we investigated the ability of PRT543 to inhibit the growth of ACC PDX tumor models in vivo and demonstrate that PRT543 significantly inhibits the growth of ACC PDX tumors in vivo. We confirmed that PRT543 downregulates levels of global symmetric dimethylarginine (sDMA, a substrate of PRMT5) in the treated tumors. Further studies with PRT543 in ex vivo and in vivo ACC models are ongoing. Collectively, these findings provide a strong molecular rationale for exploring PRT543 as a potential therapeutic option for ACC tumors. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Jack Carter, Koichi Ito, Venkat Thodima, Neha Bhagwat, Joseph Rager, Nicole Spardy Burr, Jeffrey Kaufman, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition downregulates MYB and NOTCH1 signaling, key molecular drivers of adenoid cystic carcinoma [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 1138.

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.

PRMT5 (protein arginine methyltransferase 5) is a predominant Type II PRMT that catalyzes symmetric dimethylation of protein arginine residues (sDMA). PRMT5 is overexpressed in many types of cancer and plays roles in multiple essential biological processes to promote cancer growth. Previous studies have shown that PRMT5 is a critical molecule for RNA processing and pre-mRNA splicing. Mechanistically, PRMT5 directly methylates arginine residues of several splicing factors such as Small nuclear ribonucleoprotein (SNRPB and SNRPD3) and Serine and arginine rich splicing factor 1 (SRSF1), which contributes to spliceosome assembly and promotes canonical splicing of many essential genes in cancer cells. In the present study, we examined the effects of PRT543, a potent and selective PRMT5 inhibitor, on alternative splicing in uveal melanoma which frequently express hotspot mutations on Splicing factor 3b subunit 1 (SF3B1). We first confirmed that PRT543-treated MEL202 (SF3B1R625G active mutant) and MEL270 (SF3B1WT) cells show significantly increased global alternative splicing, such as increased retained intron (RI) and skipping exon (SE), determined by delta-PSI (percentage of splice-in) analysis. PRT543 downregulates SF3B1 target genes such as FBXW5, MAP3K7, MBD4 and BRD9 that are associated with increased retention of specific intron sites. Interestingly, downregulation of the SF3B1 target genes are more significant in MEL202 (SF3B1R625G) than MEL270 (SF3B1WT), indicating that PRT543 can regulate the activity of the SF3B1 gain of function mutant. Consistent with previously reported PRMT5 knockout studies in hematological cancer cells, PRT543 also downregulates expression of SRSF1 target genes such as POLD1 and PNKP through increased intron retention in primary and metastatic uveal melanoma cell lines. Furthermore, we uncover that PRT543 strikingly increases retention of a specific intron site of ATM (ex33-34), resulting in a significant reduction of ATM protein levels in PRT543-treated MEL202 and MEL270 cells. Gene set enrichment analysis (GSEA) further reveals that PRT543 significantly and specifically regulates DNA replication and repair pathways in MEL202 cells. Importantly, combining PRT543 with DNA-alkylating agents or PARP inhibitors yields a synergistic reduction in cell viability. In summary, our results suggest that PRMT5 inhibition regulates cancer-associated RNA splicing machinery and the DNA damage response, resulting in synergistic antitumor activity when combined with chemotherapy and/or PARP inhibitors, particularly in cancers with spliceosomal mutations. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Koichi Ito, Venkat Thodima, Jack Carter, Neha Bhagwat, Monisha Sivakumar, Alexander Grego, Joseph Rager, Mizue Terai, Takami Sato, Omar Abdel-Wahab, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition regulates alternative splicing and DNA damage repair pathways in SF3B1 R625G expressing uveal melanoma cells [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 1137.

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.

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 various cellular pathways including tumorigenesis. PRMT5 can methylate histones H3 and H4, thereby leading to repression of tumor suppressor genes at specific loci. PRMT5 also regulates gene expression on a global level by methylating and altering the function of transcription factors such as p53, E2F-1, NF-κB and others. Additionally, PRMT5 has been shown to interact with members of the spliceosome complex and regulate pre-mRNA processing of key target genes. In this study, we describe the in vitro and in vivo activity of PRT543, a novel, potent and selective inhibitor of PRMT5, in hematological and solid tumor models. In a scintillation proximity based radiometric assay, PRT543 inhibited the methyltransferase activity of the PRMT5/MEP50 complex with an IC50 of 10.8 nM. It was also highly selective against a panel of 36 other methyltransferases. PRT543 robustly inhibited cell proliferation in a panel of >50 cancer cell lines, representing both solid tumors and cancers of hematological origin, in a concentration-dependent manner with IC50 values ranging from 10 - 1000 nM. This anti-proliferative activity correlated with a reduction of symmetrically dimethylated SmD3 protein, a known PRMT5 substrate, demonstrating effective inhibition of PRMT5 enzymatic activity in cells. PRT543 demonstrated good oral bioavailability and favorable pharmacokinetic properties. Oral administration of this compound led to significant tumor growth inhibition in several subcutaneous mouse xenograft models including mantle cell lymphoma (Granta-519, Z-138), acute myeloid leukemia (MV4-11, SET2, HEL), small cell lung cancer (NCI-H1048) and bladder cancer (5637) at well-tolerated doses. This in vivo activity was accompanied by a corresponding decrease in symmetrically dimethylated SmD3 in tumor tissue collected from the treated animals. PRT543 was also tested in combination with other approved targeted therapies both in vitro and in vivo. Combining PRT543 with the BCL2 inhibitor, venetoclax, revealed a potent synergistic interaction in models of leukemia and lymphoma. Further ex vivo testing in genetically-defined primary patient tumor samples is ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Neha Bhagwat, Yang Zhang, Hong Lin, Min Wang, Dave Rominger, Tom Emm, Divya Chugani-Mahtani, Dimitrios Angelis, Rupa Shetty, Raul Leal, William Gowen-MacDonald, Alexander Grego, Juan Luengo, Taghi Manshouri, Friederike Pastore, Ross L. Levine, Srdan Verstovsek, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Preclinical characterization of PRT543, a potent and selective inhibitor of protein arginine methyltransferase 5 (PRMT5), with broad antitumor activity in in vitro and in vivo models [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 2915.

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.

Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.

Constitutively activated STAT3 and STAT5 are expressed in a wide variety of human malignancies including solid and hematopoietic cancers and often correlate with a poor prognosis and resistance to multiple therapies. Given the well established role of STAT3 in tumorigenesis, inhibition of Janus-activated kinase 2 (JAK2) activity might represent an attractive therapeutic approach. Using a mouse model of colitis-induced colorectal cancer, we show that a novel, orally active, selective JAK2 inhibitor, CEP-33779, induced regression of established colorectal tumors, reduced angiogenesis, and reduced proliferation of tumor cells. Histopathologic analysis confirmed reduced incidence of histologic-grade neoplasia by CEP-33779. Tumor regression correlated with inhibition of STAT3 and NF-κB (RelA/p65) activation in a CEP-33779 dose–dependent manner. In addition, the expression of proinflammatory, tumor-promoting cytokines interleukin (IL)-6 and IL-1β was strongly reduced upon JAK2 inhibition. The ability of CEP-33779 to suppress growth of colorectal tumors by inhibiting the IL-6/JAK2/STAT3 signaling suggests a potential therapeutic utility of JAK2 inhibitors in multiple tumors types, particularly those with a strong inflammatory component. Mol Cancer Ther; 11(4); 984–93. ©2012 AACR.

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.

A novel 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine scaffold has been designed as a new kinase inhibitor platform mimicking the bioactive conformation of the well-known diaminopyrimidine motif. The design, synthesis, and validation of this new pyrrolo[2,1-f][1,2,4]triazine scaffold will be described for inhibitors of anaplastic lymphoma kinase (ALK). Importantly, incorporation of appropriate potency and selectivity determinants has led to the discovery of several advanced leads that were orally efficacious in animal models of anaplastic large cell lymphoma (ALCL). A lead inhibitor (30) displaying superior efficacy was identified and in depth in vitro/in vivo characterization will be presented.

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.

Cancer of the prostate is one of the most common malignancies and the second leading cause of cancer death in men in developed countries. There is increasing evidence that cancer stem-like cells (CSCs) are implicated in CRPC disease progression and treatment resistance. Transcriptional, epigenetic and metabolic reprogramming are key features for the acquisition and maintenance of stem-like properties. Understanding the factors regulating self-renewal and survival of prostate CSCs may offer novel targets for innovative therapeutic strategies. LSD1/KDM1A is a lysine demethylase for histone and non-histone proteins and functions as transcriptional corepressor or coactivator depending on the binding partners and substrates. LSD1 is a key epigenetic modifier controlling the fate of pluripotent stem cells in several tissues. Overexpression of LSD1 is found in many human cancers and has been linked to tumorigenic and CSC-like features. The involvement of LSD1 in stem cell pluripotency and differentiation suggests that LSD1 inhibitors, such as INCB059872, might be useful to target the prostate CSC subpopulation. Here, we examined the effects of INCB059872 on the growth properties, self-renewal and tumorigenic capability of prostate CSCs derived from human cell lines and the Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG (ERG/PTEN) mice, which develop highly invasive prostate adenocarcinomas. In ex vivo tumor-sphere assays, INCB059872 significantly suppressed the growth of tumor-initiating stem-like cells isolated from prostatic tumors generated in ERG/PTEN mice. Furthermore, treatment with INCB059872 inhibited colony and tumor-sphere formation by human prostate cancer cells. Conversely, the effects on proliferation and viability of bulk tumor cells were limited and required long-term exposure to the drug. These effects were observed in multiple prostate cancer cell lines, irrespective of the distinctive genetic features and AR status. Importantly, genetic knockdown of LSD1 by siRNAs and shRNAs recapitulated the effects of INCB059872. Both transient and stable knockdown of LSD1 had limited effects on proliferation and viability of bulk tumor cells, whereas they significantly reduced growth of colony and tumor-sphere forming stem-like cancer cells. Furthermore, LSD1 knockdown drastically reduced tumor growth and tumorigenic stem-like cells in subcutaneous xenografts of human prostate cancer cells in nude mice. These results support the hypothesis that LSD1 has a major role in sustaining the stem-like and tumorigenic subpopulation in prostate tumors and its inhibition by chemical or genetic approaches prevents survival and self-renewal capability of prostate CSCs. These data suggest that INCB059872 could be a valid addition to the current treatment strategies for prostate cancer. INCB059872 is currently in phase1/2 clinical studies Citation Format: Gianluca Civenni, Giada Zoppi, Ramiro Vazquez, Dhreeraj Shinde, Alyssa Paganoni, Aleksandra Kokanovic, Sang Hyun Lee, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. INCB059872, a novel FAD-directed LSD1 Inhibitor, is active in prostate cancer models and impacts prostate cancer stem-like cells [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 1379.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological tumor that is derived from the clonal expansion of immature T-cell progenitors. Multiple genetic and epigenetic alterations are attributed to the development of malignant T cell transformation. Among these, there is supporting evidence for a role of lysine specific demethylase (LSD1) in T-ALL. Oncogenic transcription factors, such as TAL-1, Notch, and ZEB2, form a complex with LSD1 to alter gene expression in T-ALL cells. In addition, LSD1 is aberrantly expressed in ALL, including B-ALL and T-ALL. Furthermore, the overexpression of LSD1 under control of the Sca-1 promoter in transgenic mice triggered T leukemogenesis via acquisition of self-renewal activity and alteration in the differentiation program to T-cell lineages. Together with the known function of LSD1 in regulating the activity of self-renewal in hematological malignancies, these studies prompted evaluation of the efficacy of the potent, selective, and orally bioavailable FAD-directed LSD1 inhibitor, INCB059872, in preclinical models of T-ALL. Expression of LSD1 was abundant in human-T-ALL cell lines as detected by immunoblotting. In vitro, INCB059872 treatment significantly inhibited the proliferation of a subset of human T-ALL cell lines. In vivo, once daily oral administration of INCB059872 inhibited tumor growth significantly in multiple human T-ALL subcutaneous xenograft models including Molt-4, RPMI-8402, CCRF-HSB-2, and CCRF-CEM, but was ineffective against DND-41 xenografts. The anti-tumor efficacy observed with INCB059872 had no clear genetic correlation with Notch mutation status of T-ALL tumors. Combination efficacy studies of INCB059872 with standard care of agents or targeted therapeutic agents in T-ALL models are currently being evaluated. These data suggest exploring the potential clinical development of INCB059872 as a therapy for T-ALL patients. Citation Format: Melody Diamond, Yvonne Lo, Antony Chadderton, Min Ye, Valerie Roman, Michael Weber, Chunhong He, Liangxing Wu, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Sang Hyun Lee. The evaluation of INCB059872, an FAD-directed inhibitor of LSD1, in preclinical models of T-ALL [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 1893.

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.

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.

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)–ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

Tumors of the exocrine pancreas are a major cause of cancer death and have among the poorest prognosis of any malignancy. Following the "cancer stem cell hypothesis," where tumors are believed to originate in tissue specific stem cells, we screened primary ductal pancreatic carcinomas and cell lines for the expression of possible stem cell factors. We find 32/46 (70%) of primary tumors and 9/10 (90%) of cell lines express PAX6. PAX6 is a transcription factor expressed throughout the pancreatic bud during embryogenesis but not in the mature exocrine pancreas. PAX proteins have also been implicated in maintaining stem cells in a committed but undifferentiated state but a role for PAX proteins in putative pancreas stem cells is not known. We induced a pancreatic carcinoma cell line, Panc-1, to differentiate by transfecting wild-type p53 and treating the cells with differentiation agents gastrin or butyrate. This treatment induces cells to terminally differentiate into a growth-arrested cell with neurite-like processes, express the terminal differentiation marker somatostatin and downregulate PAX6. This phenotype can be replicated by directly inhibiting PAX6 expression. These data support a model where PAX proteins are aberrantly expressed in tumors and downregulation leads to differentiation.

// 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.

The spread of intra-abdominal cancers is a vexing clinical problem for which there is no widely effective treatment. We discovered previously that (2E)-3-[(4-tert-butylphenyl)sulfonyl]acrylonitrile (1) induced cancer cell apoptosis during adhesion to normal mesothelial cells which line the peritoneum. We recently demonstrated that the sulfonylacrylonitrile portion of 1 and hydrophobic aryl substitution were essential for pro-apoptotic activity in cancer cells. Here we synthesized a diverse series of analogues of 1 in order to improve the efficacy and pharmaceutical properties. Analogues and 1 were compared in their ability to cause cancer cell death during adhesion to normal mesothelial cell monolayers. Potent analogues identified in the in vitro assay were validated and found to exhibit improved inhibition of intra-abdominal cancer in two clinically relevant murine models of ovarian and pancreatic cancer spread and metastasis, highlighting their potential clinical use as an adjunct to surgical resection of cancers.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kappa B (NF kappa B) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NF kappa B gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NF kappa B signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

Context: Medullary thyroid cancer (MTC) is a cancer of the parafollicular C cells that commonly presents with an inherited or acquired RET gene mutation. There is currently no effective systemic treatment for MTC. Objective: The objective of this study was to investigate a systemic therapeutic approach to treat MTC. We studied the sensitivity of an MTC cell line and xenograft to irinotecan, alone and in combination with the tyrosine kinase inhibitor, CEP-751. Results: In TT cell culture and xenografts, irinotecan treatment was highly effective. This effect was augmented by treatment with CEP-751. Treatment of TT cell xenografts resulted in durable complete remission in 100% of the mice, with median time to recurrence of 70 d for irinotecan alone and more than 130 d for irinotecan plus CEP-751. Although irinotecan induced an S phase checkpoint arrest in TT cells, CEP-751 in combination with irinotecan resulted in a loss of this arrest. CEP-751 induced a loss in the induction of the DNA repair program marked by phospho-H2AX and the checkpoint pathway marked by the activated Chk1 pathway. Conclusions: Irinotecan treatment was highly effective in a preclinical model of human MTC, resulting in complete remission in 100% of the xenografts treated. The duration of remission was further enhanced by combination with the kinase inhibitor, CEP-751. These results suggest that irinotecan, alone or in combination, may be useful for the treatment of MTC.

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.

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

Prostate cancer is a leading cause of cancer death worldwide. Deregulated transcriptional factors and epigenetic effectors contribute to prostate cancer progression and castration resistance providing ideal targets for developing new therapeutic strategies. Bromodomain and extra-terminal (BET) proteins act transcriptional co-activators interacting with multiple co-regulatory molecules at gene promoters and enhancers. BET inhibitors (BETi) disrupt transcriptional regulatory complexes and have broad anticancer activity. INCB054329 and INCB057643 are BETi with proven pre-clinical activity in hematological and solid tumors. Both compounds are currently in clinical trials (NCT02431260 and NCT02711137) in advanced cancer patients. In this study we explored for the first time the activity of these two BETi in prostate cancer, comparing their effects in androgen-dependent and independent models in vitro and in vivo. We assessed the effect of INCB054329 and INCB057643 on cell proliferation, colony formation and tumor-sphere assays in androgen-dependent (LNCaP and VCaP) and androgen-independent (DU145, PC3, 22Rv1) cells. The in vivo antitumor activity was evaluated in mice with 22Rv1 derived subcutaneous xenografts with drugs administered orally (BID, daily for 3 weeks). INCB054329 and INCB057643 inhibited proliferation of prostate cancer cell lines. In short-term cell proliferation assays the BETi appeared more effective against androgen-dependent (VCaP and LNCaP) than androgen-independent (DU145 and PC3) cells (GI50 of ≤100 nM and ≥500 nM, respectively). 22Rv1 cells, which express androgen-independent AR splice variants, exhibited an intermediate level of sensitivity to both compounds (GI50: 150-250 nM). Interestingly, in colony and tumor-sphere forming assays all the cell lines showed substantially greater sensitivity to the BETi than in short-term assays. Notably, androgen-independent 22Rv1 cells were highly responsive with GI50 values ≤100 nM similar to those seen in androgen-sensitive LNCaP cells. Treatment of mice bearing 22Rv1 tumor xenografts with INCB054329 (50 mg/kg) and INCB057643 (3 mg/kg) led to significant inhibition of tumor growth (T/C%: 42 and 45%, respectively) and consistent reduction of tumor weight relative to vehicle-treated mice. These results provide evidence of activity of INCB054329 and INCB057643 in prostate cancer cell lines. Although short term assays suggest a preferential anti-proliferative effect in androgen sensitive prostate cancer cells, both compounds also exhibit significant activity in an androgen-independent model both in vitro and in vivo suggesting that they might represent a valid therapeutic option for treatment of castration-resistant prostate cancer. Citation Format: Ramiro Vázquez, Gianluca Civenni, Martina Marchetti, Sabrina Zadic, Phillip Liu, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. BET inhibitors INCB054329 and INCB057643 display significant activity in androgen-independent prostate cancer models [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 5080. doi:10.1158/1538-7445.AM2017-5080

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

Pancreatic cancer is expected to become the second most deadly cancer by 2030, with few effective therapeutic options available to improve patient survival. The Kras gene is mutated in over 90% of pancreatic cancers, but the Kras protein is considered to be an undruggable target. p-Erk is a downstream effector of Kras and has been shown to be essential for the progression and maintenance of pancreatic cancer. The LSL-KrasG12D/+; Pdx-1-Cre (KC) mouse model is used to study pancreatic cancer, especially cancer progression, after stimulation with an inflammatory agent, such as caerulein or LPS. Bromodomain inhibitors are a new generation 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. BET proteins regulate several genes responsible for cell growth, apoptosis and inflammation. INCB54329 is a novel, orally bioavailable BET inhibitor that is currently being investigated in Phase 1 clinical trials.INCB054329 inhibits cell growth in murine pancreatic cancer cells harboring Kras mutations, derived from a KrasG12D/+; Trp53R172H/+; Pdx-1-Cre (KPC) mouse with an IC50 values less than 100 nM. Moreover, INCB054329 reduces the levels of p-ERK 1/2 in these cells lines. When KC mice are injected with LPS to induce inflammation and pancreatitis, p-Stat3 protein levels are significantly increased, but this pro-survival protein is reduced by more than 50%, in mice that were pre-treated with INCB054329. Tumor cells can use epigenetic modulation to evade immune recognition and to shape the TME (tumor microenvironment) toward an immunosuppressive phenotype. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is a therapeutically desirable approach. Within the TME, several cytokines and chemokines play crucial roles, recruiting and regulating inflammatory cells [macrophages, T cells and Myeloid derived suppressor cells]. KC mice stimulated with LPS have higher levels of CCL2 (1947 ± 591 ng/mL) and IL-6 (2459 ± 577 pg/mL) than unstimulated mice; these increased levels of inflammatory cytokines and the subsequent immune cell infiltration can be modulated by treatment with INCB054329, with INCB054329 demonstrating >50% inhibition of both cytokines (IL-6: 1063 ± 502 pg/mL; CCL2: 713 ± 134 ng/mL). These data suggest that INCB054329 has a dual activity, targeting cancer cells and modulating the TME; both activities may prove beneficial for the treatment of pancreatic cancer. Moreover, this work suggests that additional mechanisms may underlie the beneficial effects of bromodomain inhibitors. Citation Format: Ana Sofia Leal, Karen T. Liby, Phillip Liu, Bruce Ruggeri. The selective bromodomain inhibitor, INCB054329 targets both cancer cells and the tumor microenvironment in the KC inflammatory preclinical model of ductal pancreatic cancer [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 5082. doi:10.1158/1538-7445.AM2017-5082

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

Ewing sarcoma is a rare bone cancer affecting predominantly children. The chromosomal translocation of chromosomes 11 and 22 results in the EWS/FLI gene fusion oncoprotein that is associated with ~85% of Ewing sarcoma cases. The EWS/FLI fusion protein is involved in deregulating gene expression and consequently causing cellular transformation. It was previously reported that lysine specific demethylase 1 (LSD1) regulates EWS/FLI transcriptional activity via its functional interaction through the NuRD co-repressor complex. We therefore evaluated whether inhibition of LSD1 could have anti-tumor effects in Ewing sarcomas that express the EWS/FLI fusion oncoprotein. INCB059872 is a potent, selective, and orally available FAD-directed covalent inhibitor of LSD1. To investigate the potential utility of INCB059872 in Ewing sarcoma, the A673 cell line having the characteristic chromosomal translocation was chosen as the experimental model system. INCB059872 inhibition of LSD1 did not significantly alter A673 proliferation in vitro. However, INCB059872 inhibited oncogenic transformation as determined by colony formation clonogenicity assays. NKX.2.2 was previously identified as a critical downstream target molecule of the EWS-FLI fusion oncoprotein that is required for transformation. A significant downregulation of NKX2.2 was observed in A673 cells treated with INCB059872, suggesting that INCB059872 mediates its effects through modulation of the EWS/FLI -NKX2.2 axis. Oral administration of INCB059872 significantly suppressed the growth of both A673 and SK-ES Ewing sarcoma xenografts in vivo. In addition, in vivo efficacy was evaluated in patient derived xenograft (PDX) models that were developed from relapsed tumor tissues of Ewing sarcoma patients. Notably, a subset of PDX models having EWS/FLI translocations (3/6) exhibited significant tumor growth inhibition at well-tolerated doses of INCB059872. Molecular signatures obtained from RNA-Seq data with these PDX models exhibited intrinsic differences between responders and non-responders, suggesting additional molecular or genetic variations may contribute to their sensitivity to INCB059872. Studies identifying potential candidate molecular mechanisms are underway. Together, these data suggest that INCB059872 may be therapeutically efficacious in a subset of Ewing sarcoma patients. Citation Format: Valerie Dostalik Roman, Min Ye, Huiqing Liu, Melody Diamond, Antony Chadderton, Yvonne Lo, Xuesong M. Liu, Jin Lu, Chunhong He, Liangxing Wu, Timothy Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Gregory Hollis, Peggy Scherle, Bruce Ruggeri, Sang Hyun Lee. The evaluation of INCB059872, an FAD-directed covalent inhibitor of LSD1, in preclinical models of Ewing sarcoma [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 1162. doi:10.1158/1538-7445.AM2017-1162

Combinatorial therapeutic strategies have achieved improved response rates and durability of responses in several malignancies either by selectively targeting distinct and non-overlapping oncogenic signaling pathways (e.g. PARP and phosphoinositide 3-kinase (PI3K) inhibition in subsets of breast and ovarian cancers), or alternatively, inhibiting distinct nodal points of regulation in common oncogenic signaling pathways (e.g BRaf and MEK inhibition in subsets of melanoma). Recent data suggest that deregulated epigenetic modifications may be just as significant as genetic mutations in driving cancer development and growth by inhibition of tumor suppressor activity and activation of oncogenic pathways. We therefore hypothesized that an epigenetic regulator could potentiate the efficacy of a protein kinase inhibitor to result in robust tumor growth inhibition. We previously reported that the potent and selective LSD1 inhibitor INCB059872 potently inhibited tumor growth in multiple tumor xenograft models of AML and SCLC as a single agent and in a combination with standard of care of agents. In this study, we explored the anti-tumor effect of combining INCB059872 and various signal transduction pathway inhibitors, including the PIM kinase inhibitor INCB053914, the JAK1/2 inhibitor ruxolitinib, or the PI3K delta-selective inhibitor INCB050465 in models of human hematologic malignancies. Each of these therapeutic combinations significantly inhibited tumor growth in the Molm-16 human AML xenograft model. Mechanistic studies suggested that MYC expression levels were downregulated by these combinations both in vitro and in vivo. Treatment with INCB059872 alone or in combination with signal transduction kinase inhibitors significantly downregulated cytokines levels, particularly IL-10, sCD40L, and MCP-1 in Molm-16 tumors. These data suggest that the combination of an LSD1 inhibitor and signal transduction inhibitor can co-regulate key tumor intrinsic and extrinsic pathways involved in paracrine or autocrine signaling in AML. In addition to the improved efficacy observed in AML models, the combination of INCB059872 with the PI3Kdelta inhibitor INCB050465 enhanced tumor growth inhibition in the Will-2 xenograft model (GCB subtype, double hit lymphoma), whereas the activity of these single agents were modest in this particular subtype of lymphoma. Additional mechanistic studies are ongoing to further understand the molecular bases of these observations. Taken together, these data suggest that targeting distinct epigenetic and oncogenic signaling pathways may potentiate anti-tumor efficacy and overcome intrinsic resistance mechanisms in specific hematologic malignancies. Citation Format: Sang Hyun Lee, Matthew Stubbs, Ashish Juvekar, Melody Diamond, Antony Chadderton, Robert Collins, Xiaoming Wen, Holly Koblish, Chunhong He, Liangxing Wu, Richard Wynn, Andrew Combs, Chu-Biao Xue, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. Combination of epigenetic regulation via LSD1 inhibition with signal transduction inhibitors significantly enhances anti-tumor activity 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 2032. doi:10.1158/1538-7445.AM2017-2032

Activation of the Fibroblast Growth Factor (FGF)-FGF Receptor (FGFR) signaling axis occurs in many human cancers. In preclinical models, cell lines with genetic aberrations in FGF/FGFR genes are preferentially inhibited by compounds that selectively target the FGFR kinase. INCB54828 is a potent, selective, and reversible inhibitor of FGFR1, 2 and 3 that is currently in Phase 2 clinical trials for advanced malignancies characterized by FGF-FGFR alterations. In this study, we investigated the efficacy of INCB054828 in models of triple-negative breast cancer (TNBC). FGFR1 and FGFR2 are amplified in approximately 4% and 5% of TNBC, respectively, and oncogenic fusion proteins including FGFR3-TACC3 have also been identified in some TNBC specimens. To profile the activity of INCB054828, we screened a panel of diverse TNBC cell lines that are representative of each of the four subtypes of TNBC. Three human TNBC lines MFM223, SUM185 and SUM52PE were highly sensitive to INCB054828 in viability assays. Each of these responsive cell lines has a known alteration in FGFR, whereas TNBC lines lacking any aberrations in FGF/FGFR genes were refractory to growth inhibition. Inhibition of cell viability was associated with suppression of growth promoting pathways including Ras-MAPK. To confirm this association in vivo, four PDX models of TNBC were tested: two chemo-refractory models with FGFR1 amplification (CNV = 4 and 6) and two without any known FGF/FGFR alterations. Both of the models with FGFR1 copy number gain showed a response to INCB054828 as monotherapy with 36 and 78% tumor growth inhibition that was statistically significant vs vehicle control (P Citation Format: Phillip C.C. Liu, Brian D. Lehmann, Bruce Ruggeri, Darlise DiMatteo, Johanna M. Schafer, Jin Lu, Sang Hyun Lee, Luping Lin, Timothy C. Burn, Melody Diamond, Alla Volgina, Liangxing Wu, Gregory Hollis, Reid Huber, Jennifer A. Pietenpol, Peggy Scherle. Activity of the selective FGFR 1, 2 and 3 inhibitor INCB054828 in genetically-defined models of triple-negative breast cancer [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 531. doi:10.1158/1538-7445.AM2017-531

The CCK-A (cholecystokinin-A) receptor is selectively expressed by human pancreatic adenocarcinomas, suggesting a possible role in pancreatic tumorigenesis. In animals, pancreatic CCK receptor expression varies during ontogeny and neoplastic transformation. This study examined the temporal expression of CCK receptors in human fetal, postnatal, and adult pancreas to determine whether the appearance of CCK-A receptors in pancreatic adenocarcinomas reflected oncofetal antigen or pancreatic neoantigen expression. Messenger ribonucleic acid (mRNA) was isolated from six paraffin-embedded normal pancreatic autopsy specimens ranging in age from 17 weeks postfertilization through 26 days following full-term delivery, and samples of adult human tissues, including pancreas and pancreatic adenocarcinoma. Using reverse transcription-polymerase chain reactions, CCK-B receptor mRNA was expressed in all specimens of normal fetal and postnatal human pancreas, adult pancreas, and pancreatic adenocarcinomas. CCK-A receptor mRNA was selectively expressed only in pancreatic adenocarcinomas. These data suggest that selective CCK-A receptor expression in pancreatic adenocarcinomas reflects neoantigen expression in humans.

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.

Cholecystokinin (CCK) plays an important role in pancreatic carcinogenesis. While human CCK-A and -B receptors have been fully characterized, their relative roles in human pancreatic adenocarcinoma remain unclear. Thus, expression of CCK-A and -B receptors in normal human pancreas, pancreatic adenocarcinomas, and other human extrapancreatic tissues and malignancies was examined, using reverse transcription followed by the polymerase chain reaction (RT-PCR). mRNA isolated from 15 normal pancreas specimens, 22 pancreatic adenocarcinomas, and 58 extrapancreatic tissues and tumors was subjected to RT-PCR using primers specific for human CCK-A and -B receptors. Expression of CCK-B receptors was detected in all tissues arising from pancreas and in most extrapancreatic tissues and tumors. In contrast, CCK-A receptors exhibited a more selective pattern of expression in gall bladder, intestine, brain, ovary, spleen, and thymus. Of significance, CCK-A receptors were expressed selectively in all pancreatic adenocarcinomas, but not in any normal pancreas specimens. In situ hybridization, using receptor-specific riboprobes, localized CCK-A receptor expression to ductal cells, the presumed origin of most human pancreatic adenocarcinomas. Southern blot analysis revealed no evidence of CCK-A receptor gene amplification or rearrangement in pancreatic adenocarcinomas. Because of its selective expression, the CCK-A receptor may serve as selective biomarker for pancreatic adenocarcinoma.

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.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Indeed, a screen for essential genes in the murine MLL-AF9 retroviral model of leukemia identified the epigenetic regulators BRD4 and LSD1 as potential therapeutic targets. Furthermore, independent studies have demonstrated that inhibition of BRD4 or LSD1 by small molecules induced myeloid differentiation and suppressed leukemic stem cell phenotype in AML models. Recently it has been demonstrated that combinations of inhibitors that target distinct epigenetic regulators can exhibit synergistic effects on target gene transcription and cancer cell growth. Therefore we investigated the potential combinatorial effects of a novel FAD-directed LSD1 inhibitor, INCB059872, together with the BET inhibitor, INCB054329. The effects of single agent LSD1 and BET inhibitors or their combination were assessed using human AML models in vitro and in vivo. INCB054329 as monotherapy inhibited cell proliferation and induced apoptosis in human AML cell lines, while INCB059872 induced cellular differentiation in these cell lines as determined by the induction of myeloid differentiation markers, CD86 and CD11b. The combination of INCB054329 and INCB059872 enhanced myeloid differentiation and apoptosis in human AML cell lines compared with the single agents. Interestingly, c-myc expression was down-regulated to greater extent with the combination of both compounds compared to either agent alone. Enhanced anti-tumor efficacy with favorable tolerability was observed in human AML xenograft models when both agents were administered simultaneously or sequentially, with INCB059872 dosing regimens preceding INCB054329 administration demonstrating the greatest efficacy. These ongoing studies demonstrate that concurrent inhibition of two distinct families of epigenetic regulators, BET and LSD1, is active in preclinical AML models, and provide a rationale for the clinical evaluation of this novel, epigenetic doublet therapy in AML. Citation Format: Xuesong Liu, Matthew Stubbs, Min Ye, Roberts Collins, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Yvonne Lo, Chunhong He, Liangxing Wu, Andrew Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Phillip Liu, Sang Hyun Lee. Combination of BET inhibitor INCB054329 and LSD1 inhibitor INCB059872 is synergistic for the treatment of AML in vitro and in vivo. [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 4702.

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.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Here, we describe INCB059872, a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. INCB059872 inhibited cellular proliferation and induced cellular differentiation as measured by induction of CD86 and CD11b myeloid differentiation markers in a panel of human AML cell lines and primary human AML cells ex vivo. In vivo, pharmacodynamic (PD) assays confirmed the sustained induction of CD86 in human AML xenograft models, consistent with the mechanism of FAD-directed inhibition of LSD1. Oral administration of INCB059872 significantly inhibited tumor growth of human AML xenograft models as a single agent at doses exhibiting significant PD effects in vivo. Efficacy was further evaluated in the murine retroviral MLL-AF9 disseminated leukemia model that recapitulates hallmarks of human AML. INCB059872 significantly prolonged the median survival of MLL-AF9 expressing leukemic mice compared with vehicle treated animals. Mechanistic studies demonstrated that INCB059872 induced cell differentiation of murine blast cells, reduced blast colonies, and normalized clinical hematological parameters to those of non-leukemic mice. Notably, in both human AML xenografts and the murine MLL-AF9 leukemic model, maximal efficacy could be achieved with both daily (QD) and alternative-day (QoD) dosing regimens of INCB059872, consistent with the prolonged PD effects observed. Collectively, these studies demonstrate the key role that LSD1 activity can play in preventing leukemic cell differentiation, and support the therapeutic potential of INCB059872 in the treatment of human AML. Citation Format: Sang Hyun Lee, Matthew Stubbs, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Min Ye, Yvonne Lo, Margaret Favata, Gengjie Yang, Karen Gallagher, Lynn Leffet, Chunhong He, Liangxing Wu, Alexander Margulis, Maryanne Covington, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML. [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 4712.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Intriguingly, recent studies indicate that LSD1 inhibition can reactivate an all-trans retinoic acid (ATRA)-dependent differentiation program in non-acute promyelocytic leukemia (APL) hematologic malignancies, a genetically heterogeneous group of blood cancers that normally respond poorly to ATRA therapy. In this study, we assessed the in vitro and in vivo effects of combining ATRA with INCB059872, a potent and selective FAD-directed LSD1 inhibitor, in non-APL AML models. As a single agent, INCB059872 induced differentiation of AML cells and when combined with ATRA, synergistically promoted differentiation as indicated by induction of CD86 and CD11b expression. In addition, the combination of INCB059872 and ATRA increased apoptosis in a panel of non-APL AML cell lines. Similarly, the combination significantly increased the fraction of CD86+CD11b+ cells and reduced cell viability in a panel of primary AML cells ex vivo. These effects in both human AML cell lines and human primary AML cells were observed across distinct FAB subtypes and genetic mutation profiles. Microarray profiling coupled with bioinformatic analysis of MV-4-11 cells demonstrated that the number of regulated genes related to differentiation and apoptotic pathways was markedly elevated in cells treated with the combination of LSD1 inhibition and ATRA relative to single agents. The synergistic increase in levels of myeloid lineage transcription factors GFI1, PU.1, CEBP and a decrease in levels of the oncogene c-MYC in the combination groups were validated by q-RT-PCR and western blot analyses. In vivo, the combination of INCB059872 and ATRA enhanced CD86 and CD11b induction and reduced tumor growth in the THP-1 xenograft model of AML compared with monotherapy. Similarly, oral administration of INCB059872 and ATRA in PDX mouse models markedly increased levels of CD11b+ cells in bone marrow. Collectively, these data underscore the synergy that can exist between LSD1 inhibition and retinoic acid receptor agonism, and provide a scientific rationale for the clinical evaluation of INCB059872 and ATRA in non-APL AML patients. Citation Format: Min Ye, Mike Liu, Jin Lu, Yvonne Lo, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Paul Waeltz, Melissa Ann Fischer, Chunhong He, Liangxing Wu, Xiaochuan Shan, Hong Chang, Maryanne Covington, Yanlong Li, Tim Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Michael Savona, Martin Carroll, Bruce Ruggeri, Sang Hyun Lee. The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia. [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 4696.

Members of the JAK family of nonreceptor tyrosine kinases play a critical role in the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as a leading therapeutic target for oncology, providing a rationale for the development of a selective JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure-activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-triazolo[1,5-a]pyridine scaffold are reported. Para substitution on the aryl at the C8 position of the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2 over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of JAK2.

Anaplastic lymphoma kinase (ALK) is a pro- mising therapeutic target for the treatment of cancer, supported by considerable favorable preclinical and clinical activities over the past several years and culminating in the recent FDA approval of the ALK inhibitor crizotinib. Through a series of targeted modifications on an ALK inhibitor diaminopyrimidine scaffold, our research group has driven improvements in ALK potency, kinase selectivity, and overall pharmaceutical properties. Optimization of this scaff old has led to the identification of a potent and efficacious inhibitor of ALK, 25b .As triking feature of25b over previously described ALK inhibitors is its >600-fold selectivity over insulin receptor (IR), a closely related kinase family member. Most importantly, 25b exhibited dose proportional escalation in rat compared to compound 3 which suffered dose limiting absorption preventing further advancement. Compound 25b exhibited significant in vivo antitumor efficacy when dosed orally in an ALK-positive ALCL tumor xenograft model in SCID mice, warranting further assessment in advanced preclinical models.

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.