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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.

Alterations in fibroblast growth factor receptor (FGFR) genes have been identified as potential driver oncogenes. Pharmacological targeting of FGFRs may therefore provide therapeutic benefit to selected cancer patients, and proof-of-concept has been established in early clinical trials of FGFR inhibitors. Here, we present the molecular structure and preclinical characterization of INCB054828 (pemigatinib), a novel, selective inhibitor of FGFR 1, 2, and 3, currently in phase 2 clinical trials. INCB054828 pharmacokinetics and pharmacodynamics were investigated using cell lines and tumor models, and the antitumor effect of oral INCB054828 was investigated using xenograft tumor models with genetic alterations in FGFR1, 2, or 3. Enzymatic assays with recombinant human FGFR kinases showed potent inhibition of FGFR1, 2, and 3 by INCB054828 (half maximal inhibitory concentration [IC50] 0.4, 0.5, and 1.0 nM, respectively) with weaker activity against FGFR4 (IC50 30 nM). INCB054828 selectively inhibited growth of tumor cell lines with activation of FGFR signaling compared with cell lines lacking FGFR aberrations. The preclinical pharmacokinetic profile suggests target inhibition is achievable by INCB054828 in vivo with low oral doses. INCB054828 suppressed the growth of xenografted tumor models with FGFR1, 2, or 3 alterations as monotherapy, and the combination of INCB054828 with cisplatin provided significant benefit over either single agent, with an acceptable tolerability. The preclinical data presented for INCB054828, together with preliminary clinical observations, support continued investigation in patients with FGFR alterations, such as fusions and activating mutations.

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

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

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

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.

Genetic defects of DNA damage response (DDR) pathways in cancer cells induce synthetic lethality with chemotherapy and PARP inhibitors. However, many patients with advanced cancers develop resistance to these therapies mainly due to circumvention and/or restoration of the inactivated DDR genes. In the present study, we demonstrate that pharmacological inhibition of PRMT5, the major type II protein arginine methyltransferase, epigenetically downregulates multiple genes involved in the DDR/DNA replication pathways, resulting in the sensitization of cancer cells to chemotherapy and PARP inhibition. Treatment with C220 or PRT543, potent and selective PRMT5 inhibitors, downregulates expression of DDR genes and/or proteins including BRCA1/2, ATM/ATR, CHK1/2, RAD51, POLD1/3, and PNKP in multiple types of cancer cells in vitro. Mechanistically, PRT543 promotes global alternative splicing (ΔPSI) changes, including intron retention and exon skipping. In particular, PRT543 significantly increases retained intron of POLD1 and PNKP genes, which are downstream of the spliceosome subunit SRSF1, a major protein substrate of PRMT5. Additionally, PRT543 increases intron retention of ATM and ATR genes. For BRCA1/2, CHK1/2 and RAD51, no changes in alternative splicing were observed, suggesting the involvement of other mechanisms such as the CLNS1A/H4R3me2s chromatin regulation pathway. The regulation of the DDR pathway by PRT543 is associated with increased DNA damage as determined by COMET and gamma H2AX analyses. Combining PRT543 with PARP inhibitors or DNA-alkylating agents demonstrates a potent synergistic interaction in both HR proficient and HR deficient cancer cell lines in vitro. Similar combination effects are observed in primary cultures of patient-derived breast cancer and high-grade serous ovarian cancer. Moreover, combination of PRT543 with PARP inhibition effectively inhibits the growth of HCC1569 CDX breast cancer in vivo. Further ex vivo and in vivo combination studies with PRT543 and PARP inhibitors or chemotherapy in genetically defined CDX and PDX models are ongoing. These studies not only reveal a novel mechanism of PRMT5 inhibition, but also suggest beneficial combination effects with other therapies, particularly in patients with tumors that are resistant to therapies that are dependent on DNA damage as their mechanism of action. 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, Jack Carter, Venkat Thodima, Youyou Zhang, Monisha Sivakumar, Mu Xu, Neha Bhagwat, Joseph Rager, Jacob Spruance, Lin Zhang, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition epigenetically regulates DNA damage response pathways in cancer cells and sensitizes to chemotherapy and PARP inhibition [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 1185.

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

Acute myelogenous leukemia (AML) is the most common acute leukemia in adults. A hematologic cancer, the disease is highly heterogeneous, with multiple subtypes. Despite advances in treatment, the long-term survival for AML remains poor and the development of novel treatments is an unmet need. Due to the highly divergent subtypes and mutation profiles in AML, the use of patient-derived models may improve drug discovery and development. To address this, we have established a short-term culture system that supports the growth of primary AML cells ex vivo to permit the evaluation and/or screening of candidate agents. Our AML bank is comprised of patient-derived specimens across a range of subtypes (which includes M1, M2, M4, M5, and others), and includes models with common mutations in FLT3 (ITD), IDH1/IDH2 and NPM. Primary AML specimens were characterized for common mutations by TruSight sequencing and for surface marker expression by flow cytometry. The ex vivo assay system was evaluated for the ability to support the survival and expansion of 22 primary AML specimens. Among these, 15 had evidence of proliferation, 5 had no net expansion, and 2 failed to survive. Extension of the culture period for up to 14 days was feasible, with most models having equal or increased cell numbers by the end of the culture period (8 of 10 evaluated). All models stably expressed CD33 throughout the assay. To verify the applicability of this system for drug testing, a standard of care agent cytarabine (ara-C) was assessed for each of the AML models. Cell growth/viability was assessed using Celltiter-Glo assay. Concentration-dependent responses to ara-C were observed across multiple models (IC50 10 nM to 150 nM), indicating a range of relatively sensitive to resistant AML models. A cohort of models were evaluated in vivo for sensitivity to ara-C. Engraftment of the systemic patient-derived xenograft AML models (into NOG or NOG-EXL mice) was evaluated in circulation by flow cytometry. When engrafted, AML-bearing mice were randomized into ara-C or vehicle control groups. Two weeks later, mice were evaluated for the presence of human CD45+CD33+ AML cells. Models that were relatively sensitive to Ara C ex vivo (IC50 < 30 nM) showed a greater in vivo response as evidenced by a 60-80% reduction in the mean circulating AML cells versus models with >100 nM IC50 values, that had little response. In conclusion, Champions offers over 30 well characterized models of AML for study in ex vivo culture and in vivo systemic xenograft models. The diversity in these AML models is reflective of patient diversity, enhancing their utility in the evaluation of novel therapeutic candidates. These data indicate the feasibility of utilization of these primary models, ex vivo as well as in vivo, for drug discovery for AML, from screening to preclinical efficacy modeling. Citation Format: Bhavna Verma, Swetha Tati, Bruce Ruggeri, Amy Wesa. Establishment of a patient-derived acute myeloid leukemia (AML) ex vivo platform for evaluation of novel therapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3710.

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.

Numerous studies have elucidated that the most pivotal functions of lysine specific demethylase-1 (LSD1) are associated with regulating normal or malignant hematopoiesis by maintaining stem cell self-renewal and regulating myeloid differentiation. In preclinical models, studies with either pharmacological inhibition or genetic knockdown of LSD1 demonstrated that LSD1 is essential for differentiation of progenitor cells during normal hematopoiesis. In the clinic, AML manifests itself via clonal expansion of abnormal differentiation and proliferation of myeloid cells and, therefore, the inhibition of LSD1 activity with small molecule inhibitors could be a promising therapeutic approach for AML. Previously, we reported upon the identification of a flavin adenine dinucleotide (FAD) directed LSD1 specific inhibitor, INCB059872, which is efficacious in preclinical mouse models utilizing human AML cell lines and primary AML cells by inducing cell differentiation as indicated by the induction of CD11b and CD86 markers. Using a larger panel of myeloid and HSC flow cytometry markers, our currents efforts expanded upon these observations to ascertain whether INCB059872 enhanced lineage commitment at hematopoietic stem cell (HSC) and/ or promoted monocytic/granulocytic differentiation of human primary AML cells ex vivo and in human systemic AML PDX models. In both human AML PDX models and human primary AML samples, INCB059872 increased myeloid differentiation with increasing populations of monocytes (CD14+) and granulocytes (CD15+). Furthermore, INCB059872 induced the differentiation of early hematopoietic progenitors, CD34+/CD38- to more committed CD34+/CD38+ multipotent/oligopotent progenitors, which in turn gave rise to lineage specific progenitors in the human AML PDX models. These studies support further exploration of INCB059872 as a promising novel epigenetic agent for AML therapy whose mechanism of action lies in part through the induction of differentiation of leukemic stem/progenitor cells to more committed hematopoietic lineages. Citation Format: Antony Chadderton, Min Ye, Melody Diamond, 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 FAD-directed LSD1 specific inhibitor, INCB059872, is a promising epigenetic agent for AML therapy by inducing differentiation of leukemic stem/progenitor 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 1888.

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.

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

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

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

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.

Recent studies have demonstrated that patients with myeloproliferative disorders (MPDs) frequently have acquired activating mutations in the JAK2 tyrosine kinase. A multikinase screen determined that lestaurtinib (formerly known as CEP-701) inhibits wild type JAK2 kinase activity with a concentration that inhibits response by 50% (IC50) of 1 nM in vitro. We hypothesized that lestaurtinib would inhibit mutant JAK2 kinase activity and suppress the growth of cells from patients with MPDs. We found that lestaurtinib inhibits the growth of HEL92.1.7 cells, which are dependent on mutant JAK2 activity for growth in vitro and in xenograft models. Erythroid cells expanded from primary CD34+ cells from patients with MPDs were inhibited by lestaurtinib at concentrations of 100 nM or more in 15 of 18 subjects, with concomitant inhibition of phosphorylation of STAT5 and other downstream effectors of JAK2. By contrast, growth of erythroid cells derived from 3 healthy controls was not significantly inhibited. These results demonstrate that lestaurtinib, in clinically achievable concentrations, inhibits proliferation and JAK2/STAT5 signaling in cells from patients with MPDs, and therefore holds promise as a therapeutic agent for patients with these disorders.

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.

Among the known angiogenic growth factors and cytokines implicated in the modulation of normal and pathological angiogenesis, the VEGF family (VEGF-A, VEGF-B, VEGF-C, VEGF-D) and their corresponding receptor tyrosine kinases [VEGFR-1 (Flt-1), VEGFR-2 (Flk-1, KDR), and VEGFR-3 (Flt-4)] play a paramount and indispensable role in regulating the multiple facets of the angiogenic and lymphangiogenic processes, as well as the induction of vascular permeability and inflammation. The receptor VEGFR-2/KDR is the principal one through which VEGFs exert their mitogenic, chemotactic, and vascular permeabilizing effects on the host vasculature. Increased expression of VEGFs by tumor cells and VEGFR-2/KDR and VEGFR-1/Flt-1 by the tumor-associated vasculature are a hallmark of a variety of human and rodent tumors in vivo and correlates with tumor growth rate, micro-vessel density/proliferation, tumor metastatic potential, and poorer patient prognosis in a variety of malignancies. Approaches to disrupting the VEGF/VEGFR signaling cascade range from biological agents (soluble receptors, anti-VEGF and anti-VEGFR-2 antibodies, and VEGF transcription inhibitors) to small molecule ATP competitive VEGFR inhibitors. Examples from this latter class that are currently in clinical development include compounds from distinct chemical classes such as: indolin-2-ones, anilinoquinazolines, anilinophthalazines, isothiazoles, indolo- and indenocarbazoles. The structure activity relationships, biochemical and pharmacological profile of optimized representatives from each of these classes constitute the subject matter of this review.

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

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

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

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

Immune checkpoint blockade has shown considerable therapeutic promise in the clinic. However, single agent activity is compromised by the presence of suppressive myeloid cells, including myeloid derived suppressor cells (MDSC), tumor associated macrophages (TAM) and polymorphonuclear (PMN) cells, in the tumor microenvironment. Epigenetic alterations can significantly contribute to the development of the immunosuppressive tumor microenvironment and recent data have suggested that combining epigenetic-based therapies with immunotherapeutic agents can lead to improved efficacy in preclinical models. Since Lysine Specific Demethylase 1 (LSD1) has been shown to play a critical role in hematopoiesis, we hypothesized that inhibition of LSD1 could have a direct effect on myeloid cell differentiation and potentially restore normal myelopoiesis in cancer patients. To test this hypothesis, we evaluated INCB059872, a potent, selective and orally available FAD-directed covalent inhibitor of LSD1 in several experimental models. In an in vitro differentiation assay, the majority of CD34+ progenitor cells were driven to a monocytic phenotype in the presence of INCB059872, while control treated cells differentiated toward granulocytic PMN cells. Similar results were observed in vivo. Using the orthotopic 4T1 mammary cancer model, the myeloid compartment was characterized in tumor tissues following treatment with INCB059872. Notably, the population of PMN-MDSC was significantly decreased in tumor tissues following oral administration of INCB059872, whereas the macrophage population was increased. These data suggest that INCB059872 can redirect myeloid differentiation toward monocyte/macrophages and inhibit the differentiation of PMN-MDSC in this syngeneic tumor microenvironment. Consistently, intratumoral T lymphocyte infiltration was increased following INCB059872 treatment. The combination of INCB059872 and α-PD-L1 antibody enhanced anti-tumor efficacy in the 4T1 orthotopic tumor model. Collectively, these data suggest that inhibition of LSD1 with INCB059872 can directly affect myeloid differentiation to reduce the accumulation of myeloid suppressive cells, restoring the tumor microenvironment to be more responsive to PD-1/PD-L1 axis blockade. This study supports the therapeutic potential for the combination of an LSD1 inhibitor with immuno-therapeutic agents to improve overall clinical response in cancer patients. Citation Format: Thomas Condamine, Steve Wang, Melody Diamond, Leslie Hall, Huiqing Liu, Antony Chadderton, Jin Lu, Chunhong He, Liangxing Wu, Timothy Burn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle, Holly Koblish, Sang Hyun Lee. The LSD1 Specific Inhibitor INCB059872 enhances the activity of immune checkpoint blockade by reshaping the myeloid compartment in the syngeneic 4T1 mouse mammary tumor model [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 4635. doi:10.1158/1538-7445.AM2017-4635

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.