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Targeted protein degradation (TPD) is an emerging therapeutic modality with the potential to target previously undruggable targets. However, it has been more challenging to identify orally bioavailable TPD molecules due to the physicochemical properties of the large molecules and narrow structure-activity relationship (SAR) compared to conventional small molecule inhibitors. In the present study, we identified orally active TPD molecules that selectively and potently degrade SMARCA2 protein and induce synthetic lethality in SMARCA4-deficient cancer cells. SMARCA2 (BRM) and SMARCA4 (BRG1) are the two mutually exclusive catalytic core subunits of SWI/SNF complexes that play an important role in controlling gene expression by remodeling chromatin. The complexes are mutated in more than 20% of human cancers and subsets of solid tumors lose expression of SMARCA4 protein due to damaging mutations or gene deletion. The SMARCA4-deficient cancer cells are highly dependent on the paralog gene SMARCA2 for their survival and thus SMARCA2 has been suggested as an attractive therapeutic target for patients with SMARCA4-deficient cancers. We have recently identified SMARCA2 selective degraders that demonstrate oral bioavailability in mice with favorable pharmacokinetic properties, and acceptable DMPK and safety profiles in rodent studies. These SMARCA2 degraders show 50 to 300-fold DC50 selectivity for SMARCA2 over SMARCA4 in our cellular assays. When pre-treated with a proteasome inhibitor or neddylation inhibitor, the degradation of SMARCA2 was rescued, confirming that the degradation is mediated by the ubiquitin-proteasome-dependent pathway. These TPD molecules inhibit only SMARCA4-deficient cancer cell proliferation (NCI-H838, NCI-H1693, HT1080 SMARCA4 KO) with IC50 values ranging from 3-10 nM, but not SMARCA4 WT cells (Calu-6, NCI-H520, HT1080 WT). Oral administration of our SMARCA2 degraders resulted in significant tumor growth inhibition of SMARCA4-deficient lung cancer xenografts at well tolerated doses. The treated tumor tissues show robust SMARCA2 protein reduction for more than 72h post dosing, consistent with the SMARCA2 degradation kinetics-based pharmacodynamic prediction model. In summary, our orally bioavailable SMARCA2 degraders induce synthetic lethality in SMARCA4-deficient cancers in vitro and in vivo. Efforts to further evaluate these compounds in additional models and in combination with other agents are ongoing. Citation Format: Koichi Ito, Artem Shvartsbart, Joseph Rager, Anjana Agarwal, Michael Hulse, Komali Vykuntam, Min Wang, Justin Kurian, Miles Cowart, Joy Cote, Monisha Sivakumar, Jack Carter, Jessica Burtell, Alex Grego, Andrew Moore, Neha Bhagwat, Stefan Ruepp, Tom Emme, Liang Lu, Philip Pitis, Corey Basch, Klare Bersch, Song Mei, Raul Leal, John Rose, Danielle Roth, Ganfeng Cao, Kris Vaddi, Sandy Geeganage, Bruce Ruggeri, Andrew Combs, Peggy Scherle. Selective and orally bioavailable SMARCA2 targeted degraders induce synthetic lethality in SMARCA4- deficient solid tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6277.

Cell cycle deregulation is a hallmark of cancer and the hyperactivation and overexpression of CDKs are often drivers of cancer pathogenesis. Cyclin-dependent kinase 4 and 6 (CDK4)/(CDK6) are critical mediators of cellular transition into S phase and important for the initiation, growth, and survival of many cancers. Activated CDK4/CDK6 complexes phosphorylate Rb1, reduce their binding affinities and release Rb1-containing transcription repressor complexes from E2F transcription factors, resulting in activation of E2F controlled cell cycle genes and progression of the cell cycle. At present three CDK4/CDK6 inhibitors are approved for the treatment of ER+/HER2- breast cancer, and are being explored in other cancer indications as well. Previously we described a novel brain penetrant CDK4/CDK6 inhibitor, PRT3645, that exhibits single digit nanomolar biochemical potency against CDK4/CDK6 and >2000-fold selectivity against CDK1, CDK2 and CDK9. PRT3645 inhibits cellular phosphorylation of Rb and exhibits a protein binding-adjusted cellular IC50 of Citation Format: Yue Zou, Srijita Dhar, Kirsten Gallagher, Andrew Buesking, Sarah Pawley, Ryan Holmes, Xiaowei Wu, Katarina Rohlfing, Min Wang, Joseph Rager, Tom Emm, Stefan Ruepp, Miles Cowart, Jing Ni, Jean Zhao, Bruce Ruggeri, Andrew Combs, Kris Vaddi, Sandy Geeganage, Ashish Juvekar, Sang Hyun Lee, Peggy Scherle. The brain penetrant CDK4/6 Inhibitor, PRT3645, is highly effective in combination with other targeted therapies in preclinical models of NSCLC, CRC, and HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5973.

Adenoid Cystic Carcinoma (ACC) is a rare but aggressive malignancy of salivary gland, associated with protracted clinical course and fatal outcome. Treatment modalities are restricted to surgery and/or adjuvant radiotherapy and patients develop recurrence and distant metastasis over time. The absence of effective systematic therapy makes it incurable in advanced stage. ACC display an overall low mutation frequency, therefore very few actionable genetic alterations critical to the ACC development have been recognized. MYB fusion/overexpression is the most frequently found genetic alteration in ACC and is present in ~70% patients. This is followed by activating NOTCH mutations, reported in ~20% patients. Despite the presence of few targetable genetic alterations, recent studies revealed substantial transcriptomic heterogeneity amongst ACC tumors suggesting the role of epigenetic alterations in ACC oncogenesis. Symmetric dimethylation is an important epigenetic mechanism that regulates mRNA splicing, transcription, translation, cell cycle and oncogenic signaling pathways. Protein Arginine Methyl Transferase 5 (PRMT5) is a predominant enzyme for symmetric dimethylation among a family of 9 methyl transferases. In was suggested that PRMT5 plays a role in regulating tumor progression via modulation of MYC signaling, cancer stemness, and a wide array of additional cellular and transcriptional pro-oncogenic processes. While inhibition of PRMTs showed anti-oncogenic response in several preclinical tumor models and a subset of patients with advanced solid malignancies (including ACC), preclinical studies investigating the effect of PRMT5 blockade in ACC remain inadequate. In part, due to the scarcity of ACC specimens and limited availability of the experimental models. PRT543 is a selective and potent small molecule PRMT5 inhibitor that specifically targets PRMT5 among 37 methyl transferases. In the present study, we have investigated the effect this novel agent using a unique collection of ACC cell lines, organoids, and patient derived xenograft mouse models. To our knowledge, this is the first study investigating the therapeutic effect of PRT543 in several preclinical models of ACC. Specifically, we found that PRT543 has potent antitumor activity in in-vitro and in-vivo models with MYB expression and activating NOTCH mutations. Further, based on these observations, we have sequenced a collection of 50 ACC tumor samples to identify the subset of patients who may potentially benefit from PRT543 treatment based on their underlying genetic signatures. This study provides evidence underscoring the role of PRMT5 signaling in ACC and supports the clinical development of PRMT5 inhibitors for this indication. Citation Format: Vasudha Mishra, Alka Singh, Xiangying Chen, Michael Korzinkin, Claudia Wing, Viktoria Sarkisova, Alexandra Ozerova, Oksana Glushchenko, Venkat Thodima, Koichi Ito, Peggy Scherle, Mark Lingen, Rifat Hasina, Alexander Pearson, Ari Rosenberg, Alex Zhavoronkov, Bruce Ruggeri, Nishant Agrawal, Evgeny Izumchenko. PRT543, a methyl transferase inhibitor, has potent anti-tumor activity against adenoid cystic carcinoma of salivary glands. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4897.

SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes, containing an ATPase domain and a DNA binding bromodomain. SMARCA4 protein expression is lost in some cancers due to nonsense mutations, and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers using selective SMARCA2 degraders induces synthetic lethality while sparing SMARCA4 wild type (WT) normal cells. We have identified PRT3789, a potent and selective SMARCA2 targeted degrader, that selectively inhibits proliferation of SMARCA4-deleted cancer cells. Here, we describe the potential mechanism of action for PRT3789 at the molecular level and the in vitro and in vivo anti-tumor activity in SMARCA4-deleted cancer cells. To further elucidate the SMARCA2 degradation selectivity of PRT3789, we performed mass spectrometry to identify the selective SMARCA2 lysine residues ubiquitinated following treatment with PRT3789. This data, in combination with site-directed mutagenesis against these SMARCA2-specific ubiquitinated residues, has revealed important insights into the mechanism of action of PRT3789. In addition, to further understand the specific vulnerability of SMARCA2 in SMARCA4-deleted cells, we investigated whether PRT3789 affected the integrity of the residual SWI/SNF complex. Coimmunoprecipitation of SMARCC1 revealed that PRT3789 disrupts specific SWI/SNF complex subunits, including ACTL6A (BAF53). Functional genome-wide experiments are ongoing to evaluate the impact of this finding and the residual activity of the SWI/SNF complex. Furthermore, treatment with PRT3789 demonstrated robust inhibition of cell proliferation of SMARCA4-deleted non-small cell lung cancer (NSCLC) cells in vitro and NSCLC PDX tumors ex vivo, but not SMARCA4 WT cancer cells, in a concentration-dependent manner. Lastly, PRT3789 shows favorable pharmacokinetic properties in vivo, which correlate to its pharmacodynamics effects as evidenced by reduced SMARCA2 protein and KRT80 mRNA levels in tumor tissues. In subcutaneous cell-line derived xenograft (CDX) models of NSCLC, administration of PRT3789 demonstrated significant dose-related inhibition of SMARCA4-deleted NSCLC growth at tolerated doses, but no effect on the growth of SMARCA4 WT cancers. In summary, consistent with our previous validation studies and genomic perturbation analyses, our potent and selective SMARCA2 targeted degrader PRT3789 induces strong synthetic lethality in SMARCA4-deleted cancers in vitro and in vivo. Citation Format: Michael Hulse, Anjana Agarwal, Min Wang, Jack Carter, Monisha Sivakumar, Brian Vidal, Justin Brown, Andrew Moore, Alexander Grego, Neha Bhagwat, Joseph Rager, Liang Lu, Corey Basch, Klare Bersch, Chaofeng Dai, Philip Pitis, Andrew Combs, Bruce Ruggeri, Kris Vaddi, Peggy Scherle, Koichi Ito. Preclinical characterization of PRT3789, a potent and selective SMARCA2 targeted degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3263.

Cell cycle deregulation is a hallmark of cancer and CDK inhibitors, specifically inhibiting CDK4/6 and blocking cells transition from the G1 to the S phase of the cell cycle are the first and only class of highly specific CDK inhibitors approved for cancer treatment to date. CDK4/6 inhibitors have transformed the treatment paradigm of estrogen receptor-positive (ER+), HER2- breast cancer with three CDK4/6 inhibitors currently FDA approved. Brain metastasis commonly arises in patients with breast, lung, melanoma and other cancer types, is associated with poor survival outcomes and poses distinct challenges in clinical management. Due to advances in imaging technologies, the detection of brain metastases is increasing and there is a dearth of novel therapies to combat brain metastatic cancers and impact patient survival. Here, we describe a novel brain penetrant CDK4/6 inhibitor, PRT3645 exhibiting single digit nanomolar biochemical potency against CDK 4/6 and >2000-fold selectivity against other CDK family members (CDK1, CDK2, and CDK9). In cellular assays, PRT3645 inhibits cellular phosphorylation of RB with low nanomolar activity. Consistent with this, PRT3645 treatment resulted in concentration-dependent inhibition of cell proliferation in glioblastoma (GBM) cell lines and in HER2- and HER2+ breast cancer lines (EC50 values < 125 nM). Furthermore, PRT3645 demonstrated additive in vitro activity with fulvestrant or tucatinib in ER+ and HER2+ breast cancer lines. PRT3645 exhibits favorable in vitro safety pharmacology and ADME profiles, including brain exposure in rodents at steady state, and demonstrates oral bioavailability across rodents, dog and nonhuman primates. In vivo, oral PRT3645 was well tolerated and highly efficacious in a dose-dependent manner in subcutaneous xenograft models of GBM and breast cancer and in orthotopic human breast cancer brain metastasis (BCBM) and GBM models in mice as a monotherapy. PRT3645 showed tumor regression as single agent in the MCF7 ER+ breast cancer model and a combinatorial benefit with the estrogen receptor blocker, fulvestrant. In a HER2+ BT474-luc orthotopic model, similarly efficacious single agent activity of PRT3645 was achieved, as well as a significant combinatorial benefit on tumor growth and median survival when administered with the brain penetrant HER2 kinase inhibitor, tucatinib. PRT3645 was highly efficacious in a U87-luc GBM orthotopic model and demonstrated enhanced median survival benefit when combined with an orally active brain penetrant PRMT5 inhibitor. In summary, PRT3645 demonstrates an excellent balance of potency, selectivity, PK parameters across species, brain penetrance and favorable tissue distribution relative to brain exposure, and currently has advanced into IND-enabling preclinical studies. Citation Format: Ashish Juvekar, Yang Zhang, Andrew Buesking, Min Wang, Dave Rominger, Joseph Rager, Stefan Ruepp, Kirsten Gallagher, Yue Zou, Miles Cowart, Xiaowei Wu, Sarah Pawley, Ryan Holmes, William Gowen-MacDonald, Kris Vaddi, Andrew Combs, Bruce Ruggeri, Peggy Scherle. Brain penetrant CDK4/6 inhibitor PRT3645 demonstrates anti-tumor activity and enhances survival in glioblastoma and breast cancer brain metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2300.

PRMT5 (protein arginine methyltransferase 5) is a major Type II PRMT, which catalyzes the symmetric dimethylation of protein arginine residues (sDMA). As an epigenetic regulator, PRMT5 plays essential roles in promoting cancer growth and survival, including through mechanisms that control alternative splicing and RNA processing, and the expression of DNA damage repair genes. Spliceosome mutations have been suggested to represent a potential biomarker for PRMT5 inhibitors, and our previous work highlighted this sensitivity in SF3B1R625C/G expressing uveal melanoma cells. Here, we highlight in vitro and in vivo activity of PRT543, a potent, selective, and orally available PRMT5 inhibitor, in cancer cells harboring mutations in other spliceosome factors such as U2 small nuclear RNA auxiliary factor 1 (U2AF1) and RNA binding motif protein 10 (RBM10). Mutations in U2AF1 (including S34F hotspot mutations) and RBM10 (primarily loss of function (LOF) mutations) occur in 5-10% of all non-small cell lung cancers (NSCLC). Cell proliferation (10-day assay) was assessed in a panel of NSCLC cell lines treated with PRT543, either wild-type or harboring U2AF1S34F or RBM10LOF mutations. Strikingly, both U2AF1S34F and RBM10LOF cell lines were significantly more sensitive to PRT543 compared to wild-type cell lines. Furthermore, PRT543 induced significant dose-related tumor growth inhibition at well-tolerated doses in cell-line derived xenograft (CDX) models harboring the U2AF1S34F or RBM10LOF mutation. Consistent with our previous findings in other tumor types, PRT543 decreased expression of DNA damage repair-associated genes (e.g. BRCA1, RAD51AP1, FANCA, and FANCL) in U2AF1S34F or RBM10LOF mutant NSCLC cells. Combination with PRT543 increased the effectiveness of specific chemotherapeutic agents in both in vitro and in vivo (CDX) models of U2AF1S34F and RBM10LOF NSCLC. Efficacy studies in patient-derived xenograft (PDX) models, as well as genomic profiling of spliceosome-mutant cellular models in response to PRT543 are ongoing. 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, Monisha Sivakumar, Michael Hulse, Joseph Rager, Komali Vykuntam, Neha Bhagwat, Kris Vaddi, Bruce Ruggeri, Peggy Scherle. PRMT5 inhibitor PRT543 displays potent antitumor activity in U2AF1S34F and RBM10LOF spliceosome-mutant non-small cell lung cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2159.

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. MCL1 amplifications occur frequently in multiple tumor types. It has also been implicated in mediating resistance to chemotherapeutic agents and targeted therapies. We have previously described a novel, potent and orally bioavailable MCL1 inhibitor, PRT1419, that demonstrates anti-tumor efficacy in various preclinical models of cancer and is currently under evaluation in a Phase I clinical trial in patients with relapsed/refractory hematologic malignancies and advanced solid tumors. In an effort to identify novel biomarkers that might predict sensitivity to MCL1 inhibition, we conducted a gene dependency analysis using publicly available human cancer cell line data generated from genome-wide CRISPR/Cas9-mediated cell viability screens. We observed that mutations in the SWI/SNF complex, particularly in lung and ovarian cancer cell lines, conferred a strong functional dependency on MCL1. The mammalian SWI/SNF complex functions as a tumor suppressor in a number of cancers and regulates gene expression via chromatin-remodeling. It is comprised of multiple subunits, including one of two catalytic ATPases (SMARCA2 or SMARAC4), DNA-binding proteins ARID1A, ARID1B and ARID2, and other chromatin-binding subunits. Gene mutations in members of this complex occur in >20% of human cancers, and therapeutic agents targeting its function are under active clinical investigation. We and others have shown potent synthetic lethality with the use of SMARCA2 targeted protein degraders in SMARCA4 deleted lung cancer models. A previously published genome-wide CRISPR screen in SMARCA4-mut lung cancer cell lines demonstrated that loss of MCL1 could sensitize these cells to SMARCA2 degradation. Therefore, we evaluated PRT1419 in combination with a novel and selective SMARCA2 degrader, PRT3789, in SMARCA4 deleted lung cancer models. We observed a potent synergistic interaction in SMARCA4 deleted cell lines in vitro, whereas no additive benefit was seen in SMARCA4 WT lines. Further, combining PRT1419 and PRT3789 in vivo in cell line-derived xenograft models resulted in significant tumor growth inhibition, including tumor regressions. Additionally, we profiled PRT1419 ex vivo in a panel of lung cancer PDX models and observed significant, dose-dependent effects on cell viability in SMARCA4 deleted models with low SMARCA2 expression. In a broader lung cancer cell line viability screen conducted with PRT1419, we observed that the presence of multiple, co-occurring alterations in SWI/SNF family members such as SMARCA4, ARID1A/B mutations and loss of SMARCA2 protein were associated with sensitivity to PRT1419. Based on these findings, preclinical evaluation of PRT1419 in other tumor types with recurrent SWI/SNF mutations is ongoing. Citation Format: Norman Fultang, Neha Bhagwat, Diane Heiser, Alexander Grego, Michael Hulse, Venkat Thodima, Koichi Ito, Kris Vaddi, Bruce Ruggeri, Peggy Scherle. Combination of the MCL1 inhibitor PRT1419 and SMARCA2 degrader PRT3789 shows combinatorial benefit in SMARCA4 deleted lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 420.

CDK9 is a serine/threonine kinase belonging to the subclass of the transcription associated CDKs. CDK9 complexes with cyclin T and cyclin K, the positive transcription elongation factor b (P-TEFb), and phosphorylates Serine 2 of RNA polymerase II (pSer2 RNAPII) to activate transcription. Consequently, targeting CDK9 could effectively interfere with epigenetic and transcriptional reprogramming and prevent disease progression and treatment resistance in human cancers. Androgen receptor (AR)-dependence in prostate cancer is linked to CDK9 function. CDK9 stabilizes AR-associated proteins, and pharmacological inhibition of CDK9 can inhibit AR, AR variants, and their downstream transcription programs. We evaluated the novel CDK9 inhibitor, PRT2527, in prostate cancer models to evaluate its effects on cell proliferation, stem-like tumor cells, and tumor growth. PRT2527 is a potent inhibitor of CDK9/CyclinT1 complex, and when evaluated at concentration 200 times the biochemical IC50, PRT2527 was highly selective for CDK9 inhibition. We verified in biochemical assays the ability of PRT2527 to suppress pSer2 RNAPII and reduce expression of c-Myc, a common target of CDK9, in a concentration-dependent manner in multiple human prostate cancer cell lines. PRT2527 also inhibited c-Myc-dependent transcription in vitro in luciferase reporter assays. Furthermore, RNA sequencing showed altered expression of several genes with significant enrichment of c-Myc and E2F targets and RNAPII dependent transcription among downregulated genes in PRT2527-treated VCaP cells. In vitro, PRT2527 inhibited the proliferation of androgen-dependent and androgen-independent prostate cancer cell lines (IC50, ≤50 nM). PRT2527 was highly effective (IC50, ≤10 nM) in tumor-spheroid assays in blocking the growth of stem-like tumor cells and significantly suppressed the in vitro growth of tumor organoids from both human cell lines and patient-derived xenografts (PDXs). In mice, PRT2527 IV administration reduced pSer2 RNAPII in tumor xenografts and c-Myc-dependent transcriptional activity in a DU145 luminescence reporter model. PRT2527 administration in mice significantly reduced growth of PDX LuCaP 35 (castration-sensitive, adenocarcinoma) and LuCaP 145.2 (castration-resistant, neuroendocrine) along with the fraction of tumor-initiating stem-like cells in ex vivo assays. PRT2527 reduced pSer2 RNAPII in both PDXs, whereas c-Myc decreased in LuCaP 35 and Sox2 in LuCaP 145.2, relative to basal expression levels. Collectively, our data demonstrate that PRT2527 has potent pharmacodynamic and antitumor activity in multiple models of castration-sensitive and castration-resistant prostate cancer. PRT2527 is advancing into phase 1 studies in solid tumors. Citation Format: Elisa Federici, Gianluca Civenni, Aleksandra Kokanovic, Giada Sandrini, Luca Guarrera, Simone Mosole, Alessia Cacciatore, Valeria Uboldi, Manuel Lessi, Giovanni Papa, Domenico Albino, Elisa Storelli, Jessica Merulla, Andrea Rinaldi, Marco Bolis, Yang Zhang, Kris Vaddi, Peggy Scherle, Bruce Ruggeri, Giuseppina M. Carbone, Carlo V. Catapano. PRT2527, a novel highly selective cyclin-dependent kinase 9 (CDK9) inhibitor, is active in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5471.

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

Adenoid cystic carcinoma (ACC) is a rare cancer of secretory glands, characterized by slow and unpredictable growth, unrelenting relapse and high rates of metastasis. Poor response to chemotherapy and targeted drugs has resulted in there being no approved therapies to date, which contributes to poor prognosis with less than a 20% 5-year survival rate in patients with high-risk ACC. A vast majority of ACC tumors are known to have recurrent chromosomal translocation t(6;9) or t(8;9) resulting in MYB/MYBL1-NFIB fusions, and high levels of MYB protein expression. Furthermore, a subset of ACC tumors expresses NOTCH1 activating mutations (20-25%) and hyperactive NOTCH signaling in both primary and recurrent/metastatic tumors. A previous report has demonstrated that an inhibitor of PRMT5 (protein arginine methyltransferase) shows favorable responsiveness in patients with advanced ACC in a phase I clinical trial. Here, we investigate mechanistic aspects as to how PRMT5 inhibition potentially regulates the unique molecular drivers of ACC tumor growth. Due to a limited number of validated in vitro cellular models, we selected a group of head and neck cancer cell lines, including those originating from salivary gland, along with MYB and NOTCH1-driven leukemia models, to evaluate the effects of PRMT5 inhibition on MYB and NOTCH regulated genes. Treatment with PRT543, a potent and selective PRMT5 inhibitor, decreased expression of MYB and its associated downstream gene GATA3, as well as ACC-related genes such as SOX4 and POU3F2. Furthermore, PRT543 downregulated MYB signaling (MYB, FOXM1, NIK and SKI) as well as MYB alternative isoforms MYB-9A and MYB-10A in MYB expressing leukemia cells. Intriguingly, treatment with PRT543 also downregulated the transcription factor, HSF4, that is uniquely linked to regulation by N-terminal truncated MYB, a variant driven by an alternative promoter (TSS2) and reported as being active in ACC tumors. In addition to regulation of MYB, we also show that PRT543 downregulates NOTCH1 expression as well as multiple NOTCH1 target genes in leukemia cells which harbor a NOTCH1 active mutation. Given these results, we investigated the ability of PRT543 to inhibit the growth of ACC PDX tumor models in vivo and demonstrate that PRT543 significantly inhibits the growth of ACC PDX tumors in vivo. We confirmed that PRT543 downregulates levels of global symmetric dimethylarginine (sDMA, a substrate of PRMT5) in the treated tumors. Further studies with PRT543 in ex vivo and in vivo ACC models are ongoing. Collectively, these findings provide a strong molecular rationale for exploring PRT543 as a potential therapeutic option for ACC tumors. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Jack Carter, Koichi Ito, Venkat Thodima, Neha Bhagwat, Joseph Rager, Nicole Spardy Burr, Jeffrey Kaufman, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition downregulates MYB and NOTCH1 signaling, key molecular drivers of adenoid cystic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1138.

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

PRMT5 (protein arginine methyltransferase 5) is a predominant Type II PRMT that catalyzes symmetric dimethylation of protein arginine residues (sDMA). PRMT5 is overexpressed in many types of cancer and plays roles in multiple essential biological processes to promote cancer growth. Previous studies have shown that PRMT5 is a critical molecule for RNA processing and pre-mRNA splicing. Mechanistically, PRMT5 directly methylates arginine residues of several splicing factors such as Small nuclear ribonucleoprotein (SNRPB and SNRPD3) and Serine and arginine rich splicing factor 1 (SRSF1), which contributes to spliceosome assembly and promotes canonical splicing of many essential genes in cancer cells. In the present study, we examined the effects of PRT543, a potent and selective PRMT5 inhibitor, on alternative splicing in uveal melanoma which frequently express hotspot mutations on Splicing factor 3b subunit 1 (SF3B1). We first confirmed that PRT543-treated MEL202 (SF3B1R625G active mutant) and MEL270 (SF3B1WT) cells show significantly increased global alternative splicing, such as increased retained intron (RI) and skipping exon (SE), determined by delta-PSI (percentage of splice-in) analysis. PRT543 downregulates SF3B1 target genes such as FBXW5, MAP3K7, MBD4 and BRD9 that are associated with increased retention of specific intron sites. Interestingly, downregulation of the SF3B1 target genes are more significant in MEL202 (SF3B1R625G) than MEL270 (SF3B1WT), indicating that PRT543 can regulate the activity of the SF3B1 gain of function mutant. Consistent with previously reported PRMT5 knockout studies in hematological cancer cells, PRT543 also downregulates expression of SRSF1 target genes such as POLD1 and PNKP through increased intron retention in primary and metastatic uveal melanoma cell lines. Furthermore, we uncover that PRT543 strikingly increases retention of a specific intron site of ATM (ex33-34), resulting in a significant reduction of ATM protein levels in PRT543-treated MEL202 and MEL270 cells. Gene set enrichment analysis (GSEA) further reveals that PRT543 significantly and specifically regulates DNA replication and repair pathways in MEL202 cells. Importantly, combining PRT543 with DNA-alkylating agents or PARP inhibitors yields a synergistic reduction in cell viability. In summary, our results suggest that PRMT5 inhibition regulates cancer-associated RNA splicing machinery and the DNA damage response, resulting in synergistic antitumor activity when combined with chemotherapy and/or PARP inhibitors, particularly in cancers with spliceosomal mutations. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Koichi Ito, Venkat Thodima, Jack Carter, Neha Bhagwat, Monisha Sivakumar, Alexander Grego, Joseph Rager, Mizue Terai, Takami Sato, Omar Abdel-Wahab, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRMT5 inhibition regulates alternative splicing and DNA damage repair pathways in SF3B1 R625G expressing uveal melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1137.

SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core subunits of the SWI/SNF complexes which contain ATPase domain and DNA binding bromodomain. SMARCA4 protein expression is lost in some cancers due to damaging mutations (e.g. nonsense, frameshift deletion, splice site mutations) and SMARCA4-deleted cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. Therefore, targeting SMARCA2 in SMARCA4-deleted cancers through the use of selective SMARCA2 degraders induces synthetic lethality. We designed and synthesized a variety of novel SMARCA2 degraders and tested them for degradation potency and selectivity by high-throughput in-cell western blot and probed for both SMARCA2 and SMARCA4 proteins in SMARCA4 WT expressing non-small cell lung cancer (NSCLC) NCI-H520 cells. We identified a unique series of potent SMARCA2 degraders with 20-40 fold greater selectivity for SMARCA2 vs SMARCA4. Global proteomics analysis confirmed that these degraders are highly selective for SMARCA2, and demonstrate minimal degradation of any other protein except PBRM1, another bromodomain VIII family protein. Our data suggest that the greater degradation selectivity observed is a result of preferential SMARCA2 ternary complex formation and/or poly-ubiquitination processing. Global RNA expression analysis shows that treatment with SMARCA2 degraders downregulates gene signatures associated with the cell cycle, cell proliferation, apoptosis and cell adhesion in SMARCA4-deleted NSCLC NCI-H1693 cells. Specifically, we found a significant reduction of gene expression of AXL, SMAD3, MMP2, and KRT80, all of which are important factors for tumor cell signaling and invasion. Additionally, protein set enrichment analysis (PSEA) shows that SMARCA2 degrader-treated NCI-H1693 cells downregulate expression of cell cycle and DNA replication related protein signatures as well as upregulated antigen processing and presentation pathway. A cell line panel analysis for cell viability and clonogenicity demonstrated that our selective SMARCA2 degraders effectively inhibit proliferation of SMARCA4-deleted cancer cells with nanomolar potencies, but not SMARCA4 WT or SMARCA2/4 null cancer cells. Our SMARCA2 degraders also significantly suppressed the growth of patient derived SMARCA4-deleted NSCLC tumor cells in primary 3D culture assay. In summary, consistent with previous studies and genomic perturbation analyses, our potent and selective SMARCA2 degraders induce synthetic lethality in SMARCA4-deleted cancers in vitro. Citation Format: Koichi Ito, Anjana Agarwal, Philip Pitis, Min Wang, Jack Carter, Miles Cowart, Chaofeng Dai, William Gowen-MacDonald, Eliza Elliot, Brian Vidal, Jacob Spruance, Hsin-Yao Tang, Bruce Ruggeri, Liang Lu, Andrew Combs, Hong Lin, Peggy Scherle, Kris Vaddi. Potent SMARCA2 targeted degraders induce genetic synthetic lethality in SMARCA4 deleted cancer [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 1139.

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 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.

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.

Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. FGFR4 signaling contributes to the development and progression of subsets of cancer: in approximately 10 percent of hepatocellular carcinoma (HCC), genetic amplification of FGF19, encoding an endocrine FGF ligand that activates FGFR4-KLB receptors, has been reported. In models with this alteration, FGF19-FGFR4 signaling is oncogenic and antagonism of the FGF19-FGFR4 axis has been shown to be efficacious suggesting that selective targeting of FGFR4 may be an effective strategy for malignancies with FGFR4 activation. We describe the preclinical characterization of INCB062079 a potent and selective inhibitor of the FGFR4 kinase. In biochemical assays INCB062079 inhibited FGFR4 with low nM potency and exhibited at least 250-fold selectivity against other FGFR kinases and greater than 800-fold selectivity against a large kinase panel. This selectivity derives from the ability of INCB062079 to bind irreversibly to Cys552, a residue within the active site of FGFR4 that is non-conserved among other FGFR receptors. Covalent binding of INCB062079 to Cys552 was demonstrated using a LC/MS/MS-based proteomic analysis that confirmed specificity for the target Cys. In assays using HCC cells with autocrine production of FGF19, INCB062079 inhibited the autophosphorylation of FGFR4 and blocked signal transduction by FGFR4 to downstream markers of pathway activation. Cancer cell lines that have amplification and expression of FGF19 are uniquely sensitive to growth inhibition by INCB062079 (EC50 less than 200 nM) compared with HCC cell lines or normal cells without FGF19-FGFR4 dependence (EC50 > 5000 nM) confirming selectivity for FGFR4. In vivo, oral administration of INCB062079 inhibited the growth and induced significant regressions of subcutaneous xenograft tumors dependent upon FGFR4 activity at doses that were well-tolerated (10-30 mg/kg BID) and did not result in a significant increase in serum phosphate levels which is observed with FGFR1/2/3 inhibition. Suppression of tumor growth correlated with pharmacodynamic inhibition of FGFR4 signaling. Collectively, these preclinical studies demonstrate that INCB062079 potently and selectively inhibits models of FGF19-FGFR4-dependent cancers in vitro and in vivo, supporting clinical evaluation in patients harboring oncogenic FGFR4 activation. Citation Format: Phillip C.C. Liu, Liang Lu, Kevin Bowman, Matthew C. Stubbs, Liangxing Wu, Darlise DiMatteo, Sindy Condon, Ronald Klabe, Ding-Quan Qian, Xiaoming Wen, Paul Collier, Karen Gallagher, Michael Hansbury, Xin He, Bruce Ruggeri, Yan-ou Yang, Maryanne Covington, Timothy C. Burn, Sharon Diamond-Fosbenner, Richard Wynn, Reid Huber, Wenqing Yao, Swamy Yeleswaram, Peggy Scherle, Gregory Hollis. Selective inhibition of FGFR4 by INCB062079 is efficacious in models of FGF19- and FGFR4-dependent cancers [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 2100. doi:10.1158/1538-7445.AM2017-2100

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

Methylated histone marks on H3K4 and H3K9 are generally coupled with transcriptional activation and repression, respectively. Altered levels of these histone methylation marks lead to abnormal gene expression and are associated with oncogenesis. Lysine specific demethylase 1 (LSD1) catalyzes demethylation of mono and di-methylated lysine 4 and 9 of histone H3 via an FAD-dependent redox-process. Deregulated LSD1 activity perturbs normal gene expression and can lead to cellular transformation. In particular, the function of LSD1 has been reported to maintain stem cell-like gene expression patterns in various cancers, most notably small cell lung cancer (SCLC), a cancer type that is characterized by a highly undifferentiated state and with high unmet medical need. INCB059872 is a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. The effect of INCB059872 in SCLC cell lines was assessed in vitro and in vivo. In these studies, the proliferation of a panel of SCLC cell lines was inhibited by INCB059872, with EC50 values ranging from 47 to 377 nM. Non-tumorigenic cells, such as IL-2 stimulated T cells from normal donors, by contrast, were significantly less sensitive with IC50 values > 10 μM. Oral administration of INCB059872 on once daily (QD) and alternative day (QoD) dosing regimens inhibited tumor growth in the NCI-H526 and NCI-H1417 human SCLC xenograft models. Consistent with previous reports, INCB059872 treatment in these models led to the induction of the FEZ1 and UMODL1 genes relative to vehicle-treated animals, as part of a gene signature in SCLC cell lines that is predictive of LSD1 responsiveness. Moreover, serum levels of the neuroendocrine marker pro-GRP were markedly reduced at all efficacious dosing regimens in the NCI-H1417 human SCLC xenograft model, suggesting that serum pro-GRP levels could be used as a surrogate pharmacodynamic marker of LSD1 inhibition. Currently, the efficacy of INCB059872 in combination with standard of care therapies for SCLC is under evaluation in these preclinical models. These data support the clinical evaluation of INCB059872 in patients with SCLC. Citation Format: Sang Hyun Lee, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Margaret Favata, Chunhong He, Liangxing Wu, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. The evaluation of INCB059872, an FAD-directed inhibitor of LSD1, in preclinical models of human small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4704.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Indeed, a screen for essential genes in the murine MLL-AF9 retroviral model of leukemia identified the epigenetic regulators BRD4 and LSD1 as potential therapeutic targets. Furthermore, independent studies have demonstrated that inhibition of BRD4 or LSD1 by small molecules induced myeloid differentiation and suppressed leukemic stem cell phenotype in AML models. Recently it has been demonstrated that combinations of inhibitors that target distinct epigenetic regulators can exhibit synergistic effects on target gene transcription and cancer cell growth. Therefore we investigated the potential combinatorial effects of a novel FAD-directed LSD1 inhibitor, INCB059872, together with the BET inhibitor, INCB054329. The effects of single agent LSD1 and BET inhibitors or their combination were assessed using human AML models in vitro and in vivo. INCB054329 as monotherapy inhibited cell proliferation and induced apoptosis in human AML cell lines, while INCB059872 induced cellular differentiation in these cell lines as determined by the induction of myeloid differentiation markers, CD86 and CD11b. The combination of INCB054329 and INCB059872 enhanced myeloid differentiation and apoptosis in human AML cell lines compared with the single agents. Interestingly, c-myc expression was down-regulated to greater extent with the combination of both compounds compared to either agent alone. Enhanced anti-tumor efficacy with favorable tolerability was observed in human AML xenograft models when both agents were administered simultaneously or sequentially, with INCB059872 dosing regimens preceding INCB054329 administration demonstrating the greatest efficacy. These ongoing studies demonstrate that concurrent inhibition of two distinct families of epigenetic regulators, BET and LSD1, is active in preclinical AML models, and provide a rationale for the clinical evaluation of this novel, epigenetic doublet therapy in AML. Citation Format: Xuesong Liu, Matthew Stubbs, Min Ye, Roberts Collins, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Yvonne Lo, Chunhong He, Liangxing Wu, Andrew Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Phillip Liu, Sang Hyun Lee. Combination of BET inhibitor INCB054329 and LSD1 inhibitor INCB059872 is synergistic for the treatment of AML in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4702.

Inhibitors of the BET family of Bromodomain proteins have been shown to be growth inhibitory across a spectrum of tumor types due to their ability to regulate expression of key survival and cell fate determining genes such as c-myc. Among the various tumor histologies, hematologic malignancies are among the most sensitive cancers to BET inhibition. INCB054329 is a novel, non-benzodiazepine, selective BET inhibitor that is undergoing Phase 1 clinical trials and that has shown encouraging in vitro and in vivo preclinical activity in several models of hematologic malignancy. In the current study, the activity of INCB054329 was evaluated in models of B cell malignancy. INCB54329 effectively inhibited the in vitro growth of a panel of cell lines representing both Hodgkin and non-Hodgkin lymphoma. Treated cells arrested primarily in G1 with sensitive lines also exhibiting dose and time-dependent apoptosis. Within a panel of double-hit lymphoma cell lines, which have activating chromosomal rearrangements in both c-myc and bcl-2, INCB054329 potently inhibited cell growth and was more effective than antagonists of BTK, bcl-2, PIM and PI3Kδ. INCB054329 also showed in vivo efficacy in models of diffuse large B-cell lymphoma (DLBCL). As a single agent, oral administration of INCB054329 inhibited tumor growth in Pfeiffer (GBC) and WILL-2 (GCB, double-hit) subcutaneous xenograft models. The in vivo combination of bendamustine with INCB054329 enhanced anti-tumor efficacy compared with either agent alone in the Pfeiffer model, and the combination was well tolerated. A rational, targeted combination strategy was evaluated involving INCB054329 and a selective, orally active PI3Kδ inhibitor, INCB050465, which is currently in clinical trials in B cell malignancies. Combining INCB054329 with PI3Kδ inhibition markedly enhanced anti-tumor efficacy, increasing the incidence of partial tumor regressions in vivo. In this model, both INCB054329 and INCB050465 treatment led to a reduction in c-Myc protein levels, suggesting a convergence between modulation of BET transcriptional regulation and the PI3Kδ pathway. These data suggest that clinical investigation of INCB054329, both as monotherapy and in combination with standard of care or novel targeted therapies, in several classes of B cell lymphoma, including high risk double hit lymphoma, is warranted. Citation Format: Matthew Stubbs, Robert Collins, Alla Volgina, Mike Liu, Margaret Favata, Mark Rupar, Xiaomng Wen, Richard Sparks, Thomas Maduskuie, Maryanne Covington, Timothy Burn, Bruce Ruggeri, Andrew P. Combs, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip CC Liu. Activity of the BET inhibitor INCB054329 in models of lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3780.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Here, we describe INCB059872, a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. INCB059872 inhibited cellular proliferation and induced cellular differentiation as measured by induction of CD86 and CD11b myeloid differentiation markers in a panel of human AML cell lines and primary human AML cells ex vivo. In vivo, pharmacodynamic (PD) assays confirmed the sustained induction of CD86 in human AML xenograft models, consistent with the mechanism of FAD-directed inhibition of LSD1. Oral administration of INCB059872 significantly inhibited tumor growth of human AML xenograft models as a single agent at doses exhibiting significant PD effects in vivo. Efficacy was further evaluated in the murine retroviral MLL-AF9 disseminated leukemia model that recapitulates hallmarks of human AML. INCB059872 significantly prolonged the median survival of MLL-AF9 expressing leukemic mice compared with vehicle treated animals. Mechanistic studies demonstrated that INCB059872 induced cell differentiation of murine blast cells, reduced blast colonies, and normalized clinical hematological parameters to those of non-leukemic mice. Notably, in both human AML xenografts and the murine MLL-AF9 leukemic model, maximal efficacy could be achieved with both daily (QD) and alternative-day (QoD) dosing regimens of INCB059872, consistent with the prolonged PD effects observed. Collectively, these studies demonstrate the key role that LSD1 activity can play in preventing leukemic cell differentiation, and support the therapeutic potential of INCB059872 in the treatment of human AML. Citation Format: Sang Hyun Lee, Matthew Stubbs, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Min Ye, Yvonne Lo, Margaret Favata, Gengjie Yang, Karen Gallagher, Lynn Leffet, Chunhong He, Liangxing Wu, Alexander Margulis, Maryanne Covington, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4712.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Intriguingly, recent studies indicate that LSD1 inhibition can reactivate an all-trans retinoic acid (ATRA)-dependent differentiation program in non-acute promyelocytic leukemia (APL) hematologic malignancies, a genetically heterogeneous group of blood cancers that normally respond poorly to ATRA therapy. In this study, we assessed the in vitro and in vivo effects of combining ATRA with INCB059872, a potent and selective FAD-directed LSD1 inhibitor, in non-APL AML models. As a single agent, INCB059872 induced differentiation of AML cells and when combined with ATRA, synergistically promoted differentiation as indicated by induction of CD86 and CD11b expression. In addition, the combination of INCB059872 and ATRA increased apoptosis in a panel of non-APL AML cell lines. Similarly, the combination significantly increased the fraction of CD86+CD11b+ cells and reduced cell viability in a panel of primary AML cells ex vivo. These effects in both human AML cell lines and human primary AML cells were observed across distinct FAB subtypes and genetic mutation profiles. Microarray profiling coupled with bioinformatic analysis of MV-4-11 cells demonstrated that the number of regulated genes related to differentiation and apoptotic pathways was markedly elevated in cells treated with the combination of LSD1 inhibition and ATRA relative to single agents. The synergistic increase in levels of myeloid lineage transcription factors GFI1, PU.1, CEBP and a decrease in levels of the oncogene c-MYC in the combination groups were validated by q-RT-PCR and western blot analyses. In vivo, the combination of INCB059872 and ATRA enhanced CD86 and CD11b induction and reduced tumor growth in the THP-1 xenograft model of AML compared with monotherapy. Similarly, oral administration of INCB059872 and ATRA in PDX mouse models markedly increased levels of CD11b+ cells in bone marrow. Collectively, these data underscore the synergy that can exist between LSD1 inhibition and retinoic acid receptor agonism, and provide a scientific rationale for the clinical evaluation of INCB059872 and ATRA in non-APL AML patients. Citation Format: Min Ye, Mike Liu, Jin Lu, Yvonne Lo, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Paul Waeltz, Melissa Ann Fischer, Chunhong He, Liangxing Wu, Xiaochuan Shan, Hong Chang, Maryanne Covington, Yanlong Li, Tim Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Michael Savona, Martin Carroll, Bruce Ruggeri, Sang Hyun Lee. The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4696.

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

Tumor-associated angiogenesis is critical for tumor growth and metastasis and is controlled by various pro- and antiangiogenic factors. The Eph family of receptor tyrosine kinases has emerged as one of the pivotal regulators of angiogenesis. Here we report that interfering with EphA signaling resulted in a pronounced inhibition of angiogenesis in ex vivo and in vivo model systems. Administration of EphA2/Fc soluble receptors inhibited, in a dose-dependent manner, microvessel formation in rat aortic ring assay, with inhibition reaching 76% at the highest dose of 5000 ng/ml. These results were further confirmed in vivo in a porcine aortic endothelial cell-vascular endothelial growth factor (VEGF)/basic fibroblast growth factor Matrigel plug assay, in which administration of EphA2/Fc soluble receptors resulted in 81% inhibition of neovascularization. The additive effects of simultaneous inhibition of VEGF receptor 2 and EphA signaling pathways in aortic ring assay and antiangiogenic efficacy of EphA2/Fc soluble receptors against VEGF/basic fibroblast growth factor-mediated neovascularization in vivo indicated a critical and nonredundant role for EphA signaling in angiogenesis. Furthermore, in two independent experiments, we demonstrated that EphA2/Fc soluble receptors strongly (by ∼50% versus controls) suppressed growth of ASPC-1 human pancreatic tumor s.c. xenografts. Inhibition of tumor growth was due to decreased proliferation of tumor cells. In an orthotopic pancreatic ductal adenocarcinoma model in mice, suppression of EphA signaling by i.p. administration of EphA2/Fc (30 μg/dose, three times a week for 56 days) profoundly inhibited the growth of primary tumors and the development of peritoneal, lymphatic, and hepatic metastases. These data demonstrate a critical role of EphA signaling in tumor growth and metastasis and provide a strong rationale for targeting EphA2 receptors for anticancer therapies.

All of the cases of medullary thyroid carcinoma (MTC) express the RET receptor tyrosine kinase. In essentially all of the hereditary cases and approximately 40% of the sporadic cases of MTC, the RET kinase is constitutively activated by mutation. This suggests that RET may be an effective therapeutic target for treatment of MTC. We show that the indolocarbazole derivatives, CEP-701 and CEP-751, inhibit RET in MTC cells. These compounds effectively inhibit RET phosphorylation in a dose-dependent manner at concentrations100 nM in 0.5% serum and at somewhat higher concentrations in the presence of 16% serum. They also blocked the growth of these MTC cells in culture. CEP-751 and its prodrug, CEP-2563, also inhibited tumor growth in MTC cell xenografts. These results show that inhibiting RET can block the growth of MTC cells and may have a therapeutic benefit in MTC.

We describe a novel series of small molecule ATP-competitive inhibitors of the atypical class of Protein Kinase C (PKC). The PKC family of serine/threonine kinases are divided into four structurally and functionally distinct sub-types: classic (PKCα, PKCβ, PKCγ); novel (PKCδ, PKCε, PKCη, PKCθ); atypical (PKCι,PKCζ); and PKNs (PKN1, PKN2, PKN3). The PKCs have been defined as key regulators in a multitude of signal transduction pathways that impinge on diverse cellular processes such as proliferation, differentiation, and survival. The regulation and functions of the atypical PKCs are distinct from the classic and novel PKCs.Both atypical isoforms have been implicated in various models of cancer, and PKCι in particular has been described as a potential oncogene. Its abundance is frequently increased, primarily through PKCι gene amplification, in many epithelial tumours including: subsets of squamous non-small cell lung carcinoma, serous ovarian carcinoma, and squamous esophageal carcinoma. It has been widely demonstrated that deregulation of PKCι signalling leads to unconstrained cell growth, increased migratory and invasive behaviour, and aberrant cellular polarity, which is a hallmark of aggressive cancers. Together, these data make a strong case for the inhibition of PKCι as a novel therapeutic strategy. We have discovered a series of ATP-competitive thieno[3,2-d]pyrimidine- based PKCι inhibitors from a high throughput screen. Using structure-based design we have optimized the series and demonstrated potent and selective inhibition of PKCι in biochemical and cellular models. The biochemical, structural, and cellular characterization of these compounds will be described herein. Citation Format: Christelle Soudy, Emma Stanway, Bhavisha Patel, Caroline Barton, Michelle Barnard, Leon Pang, Paul Owen, Andrew Turnbull, Bruce A. Ruggeri, Bruce D. Dorsey, Mark A. Ator, Greg R. Ott, Mark Linch, Philippe Riou, Peter J. Parker, Sven Kjaer, Christian Dillon, Neil Q. McDonald, Jon Roffey. Identification and characterization of small molecule thieno[3,2-d]pyrimidine inhibitors of Protein Kinase C iota (PKCι). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-99. doi:10.1158/1538-7445.AM2014-LB-99

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

Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60-70% of melanomas, 23-83% of papillary thyroid carcinomas, 4-16% colorectal cancers, and a lesser extent in serous ovarian and non-small-cell lung cancers. The V600E mutation is found in the vast majority of these cases and is an activating mutation, conferring transforming and immortalization potential to cells. CEP-32496 is a potent BRAF inhibitor that demonstrates selective anti-tumor activity against BRAFV600E mutated cancer cells both in vitro and in vivo. CEP-32496 is orally bioavailable and demonstrates in vivo pharmacodynamic inhibition of pMEK and pERK signaling and tumor stasis and regressions in BRaf V600E cell line tumor xenograft models. In the current study Champions primary human TumorGraft models of melanoma and colorectal carcinoma possessing the B-Raf V600E genotype were utilized to determine the oral anti-tumor efficacy of CEP-32496 in more clinically relevant models in order to support the clinical development of CEP-32496. Champions Oncology has developed an innovative platform for oncology research that utilizes the implantation of primary human tumors in immune-deficient mice in a manner that preserves the biological properties of the original human tumor. Previous reports have demonstrated the correlation between the responses observed in Champions TumorGraft™ models and clinical responses from the patients from which the models were derived. CEP-32496 was evaluated in a panel of nine Champions TumorGraft models and compared to the recently FDA-approved B-RAF inhibitor, vemurafenib (Roche/Genentech/Daiichi) in order to determine the potential clinical activity of the compound. Mice bearing established Champions TumorGrafts were treated orally with 35 mg/kg bid of CEP-32496 or vemurafenib for 25 to 35 days and anti-tumor efficacy and tolerability were evaluated. Results demonstrated that in 5/9 (56% of the Tumorgraft models) CEP-32496 showed significantly superior in vivo efficacy compared to vemurafenib. Equivalent anti-tumor efficacy was observed in 3/9 (33%) TumorGraft models with both CEP-32496 and vemurafenib. Both inhibitors demonstrated comparable tolerability profiles. Additional TumorGraft models are currently under evaluation and retrospective bioinformatics analyses are planned to determine potential signatures of response and resistance for CEP-32496 to facilitate both its clinical development and potential differentiation from vemurafenib. Overall, these results demonstrate that CEP-32496 shows comparable to superior efficacy in this panel of Champions primary TumorGraft models when compared to vemurafenib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3755. doi:1538-7445.AM2012-3755

Anaplastic lymphoma kinase (ALK) was originally identified as the oncogenic NPM (nucleophosmin)-ALK fusion protein due to a t (2;5) chromosomal translocation in anaplastic large cell lymphomas (ALCL). Many other chromosomal rearrangements or gene mutations/amplification leading to enhanced ALK activity have subsequently been identified and characterized in a number of human cancer types. The recent reports of EML4 (echinoderm microtubule-associated protein-like 4)-ALK oncogenic proteins in non-small cell lung cancer (NSCLC) and the identification of ALK activating point mutations and gene amplification in neuroblastoma have indicated ALK as a potential major therapeutic target for human cancers. Here we report the identification and preclinical characterization of CEP-28122, a highly potent and selective orally active ALK inhibitor. CEP-28122 is a potent inhibitor of recombinant ALK activity (IC50 of 3 nM) and NPM-ALK tyrosine phosphorylation in ALCL cells (IC50 of 20-30 nM). It is selective over a broad panel of protein kinases and a panel of receptors and ion channels with greater than 300-fold selectivity for insulin receptor. CEP-28122 induced concentration-dependent growth inhibition and cytotoxicity of ALK-positive ALCL and NSCLC cells with minimal activity against ALK-negative lymphoma and leukemia cells as well as ALK-negative NSCLC cells at concentrations up to 3 μM. CEP-28122 exhibited favorable oral bioavailability (F = 37-71% across species) with adequate tissue distribution in rodents. It displayed dose-dependent inhibition of NPM-ALK tyrosine phosphorylation in human ALCL tumor xenografts in mice with complete target inhibition (> 90%) for more than 12 h following single oral dosing at 30 mg/kg. Dose-dependent anti-tumor activity was observed in NPM-ALK-positive Sup-M2 and Karpas-299 ALCL tumor xenografts and EML4-ALK-positive NCI-H2228 and NCI-H3122 tumor xenografts in mice dosed with CEP-28122, bid, po, with complete or near complete tumor regressions observed following 2 weeks of treatment with CEP-28122 at 30 mg/kg or higher. Treatment of mice bearing Sup-M2 tumor xenografts or primary human ALCL tumorgrafts with CEP-28122 at 55 or 100 mg/kg bid, po, for 4 weeks led to sustained tumor regression in all mice, with no tumor re-emergence in any mouse up to 60 days post cessation of CEP-28122 treatment. On the contrary, CEP-28122 displayed marginal anti-tumor activity against ALK-negative lymphoma and NSCLC tumor xenografts under the same dosing regimens. It was well tolerated with all dosing regimens in mice and rats with no overt toxicity and no compound-related body weight loss. CEP-28122 advanced into preclinical development based on its potency, selectivity and overall favorable pharmacological, pharmaceutical and safety profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2011-3574

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is the most common cancer in the world, and is lethal in 90% of the cases. In non-small cell lung cancer (NSCLC), deregulated receptor tyrosine kinases (RTKs) stand out among the causal dominant oncogenes. Consequently, the genomic and/or pharmacological ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nevertheless, this approach has serious constrains, probably due to the frequent co-activation of multiple RTKs in a considerable subsets of solid tumors. It has become also evident that during TK inhibitor-based regimens, RTK switching and/or the selection of resistant clones can frequently occur representing a serious and problematic drawback. In a subset of NSCLC, the Anaplastic Lymphoma Kinase (ALK) gene has been described to be translocated and fused to EML4. This determines the ALK constitutive dimerization and autophosphorylation leading to cellular transformation. Here, we investigated the ALK oncogenic addiction of human NSCLC and studied the putative co-operative role of other kinases. We first demonstrated that the ectopic expression of EML4-ALK in ALK positive NSCLC cell lines (H2228 and H3122) resulted in the activation of multiple signaling pathways, in manner similar to that described for other known ALK fusions. Although EML4-ALK can induce transformation in lung in vitro and in vivo, ALK inhibition via shRNA or small molecule inhibitors (CEPs, Cephalon) induced only the apoptosis of H3122 cells, whereas in H2228 it caused cell growth arrest. Moreover, the treatment with ALK inhibitors led to tumor regression, but not tumor eradication, in vivo. Based on Phosphoproteomic analyses we demonstrated that the phosphorylation status of several tyrosine kinases (such as, EGFR, Met, FGFR, Jak1 or IGFR) was affected by the ALK inhibition only in H2228 cell line, but not in H3122. Notably, the combined treatment with anti-ALK ([CEP14083][1], CEP2550, [CEP28122][2]) and -EGFR inhibitors resulted in an increased cell death of H2228 cells to values similar to those observed for ALK treated H3122 cells. Finally, gene expression analyses showed that known EGFR substrates were specifically down regulated upon the combined treatment in ALK positive H2228 cells. Our findings suggest that ALK signaling is required for the maintenance of the neoplastic phenotype of some ALK positive NSCLC cells and that its abrogation could represent a novel strategy for the treatment of a well-defined subset of human lung cancer (complete ALK addiction). More importantly, we showed that the tumor survival and maintenance of ALK positive neoplastic cells might relay on the concomitant activation of multiple RTKs. These findings further support the notion that molecular and functional signatures are required for designing molecular-based “patient specific” therapeutic protocols in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-309. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP14083&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom [2]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP28122&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom

Anaplastic large cell lymphomas (ALCLs) are a unique subgroup of high grade non-Hodgkin lymphomas. Approximately 60% to 70% of ALCLs contain a t (2:5) (p23; q35) chromosomal rearrangement, generating the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein. NPM-ALK encodes a constitutively activated tyrosine kinase directly involved in the pathogenesis of ALCL. Recent studies have provided solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK (+) ALCL cells. However, differential sensitivity to an ALK inhibitor among human NPM-ALK (+) ALCL cell lines has also been observed. In this study, using 2 fused pyrrolocarbazole (FP)-derived ALK inhibitors and a diaminopyrimidine (DAP)-derived ALK inhibitor as tools, the sensitivity of ALCL cells to ALK inhibition and the signaling pathway(s) that may contribute to the differential sensitivity of ALCL cells to ALK inhibition were characterized and compared. In all four NPM-ALK (+) ALCL cell lines tested, inhibition of Stat3 phosphorylation was consistent with ALK inhibitory activity. In contrast, inhibition of Akt phosphorylation by the FP- or DAP-ALK inhibitor was only observed in the more sensitive cell lines, Sup-M2 and Sudhl-1 cells while in less sensitive cell lines, Karpas-299 and SR-786 cells, Akt was constitutively activated even when ALK phosphorylation was completed abolished. Treatment with LY-294002, a PI3K inhibitor, at 30 µM completely inhibited Akt phosphorylation and led to partial growth inhibition of Karpas-299 and SR-786 cells. Co-treatment with LY-294002 and the FP- or DAP-ALK inhibitor significantly enhanced the cytotoxicity against Karpas-299 and SR-786 cells, similar to the degree observed in Sup-M2 or Sudhl-1 cells treated with the same concentration of ALK inhibitor alone, suggesting PI3K/Akt activity is a major determinant for the sensitivity of NPM-ALK (+) ALCL to ALK inhibition. Karpas-299 subcutaneous tumor xenografts displayed faster growth rate than Sup-M2 tumor xenografts in mice, and the DAP-ALK inhibitor was less efficacious against Karpas-299 tumor xenografts (about 40% TGI and 55% TGI respectively @ 30 or 55 mg/kg, bid, po) compared to that observed against Sup-M2 tumor xenografts (partial and near complete tumor regression respectively at the same dose regimes). Treatment of mice bearing Karpas-299 tumor xenografts with rapamycin, an mTOR inhibitor, @ 10 mg/kg, ip led to the inhibition of S6 and eIF4G phosphorylation in tumor xenografts. Co-administration of rapamycin (10 mg/kg, qd, ip) and the DAP-ALK inhibitor (55 mg/kg, bid, po) significantly enhanced the anti-tumor efficacy against Karpas-299 tumor xenografts and led to near complete tumor regression. These data further support that PI3K/Akt/mTOR activity is a major determinant for the sensitivity of NPM-ALK (+) ALCL to ALK inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2503.