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Supplementary Table from Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor

Supplementary Figure from Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies.Significance:We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach.See related commentary by Capparelli and Aplin, p. 1413.This article is highlighted in the In This Issue feature, p. 1397

Supplementary Data from Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor

Supplemental Tables 1 & 2 Supplemental Figure Legends Supplemental Figures 1 - 5

Purpose:Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials.Experimental Design:We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations.Results:In addition to c-MYC, INCB054329 decreased expression of oncogenes FGFR3 and NSD2/MMSET/WHSC1, which are deregulated in t(4;14)-rearranged cell lines. The profound suppression of FGFR3 sensitized the t(4;14)-positive cell line OPM-2 to combined treatment with a fibroblast growth factor receptor inhibitor in vivo. In addition, we show that BET inhibition across multiple myeloma cell lines resulted in suppressed interleukin (IL)-6 Janus kinase–signal transducers and activators of transcription (JAK–STAT) signaling. INCB054329 displaced binding of BRD4 to the promoter of IL6 receptor (IL6R) leading to reduced levels of IL6R and diminished signaling through STAT3. Combination with JAK inhibitors (ruxolitinib or itacitinib) further reduced JAK–STAT signaling and synergized to inhibit myeloma cell growth in vitro and in vivo. This combination potentiated tumor growth inhibition in vivo, even in the MM1.S model of myeloma that is not intrinsically sensitive to JAK inhibition alone.Conclusions:Preclinical data reveal insights into vulnerabilities created in myeloma cells by BET protein inhibition and potential strategies that can be leveraged in clinical studies to enhance the activity of INCB054329.

Background: Lung cancer is one of the most common tumors, accounting for approximately 1.8 million deaths worldwide in 2020. KRAS is a frequently mutated oncogene, with mutations reported in roughly 20-25% of non-small cell lung cancer cases. Specifically, KRAS mutations at amino acid 12, resulting in a glycine to cysteine (G12C) substitution, occur in approximately 13% and 3% of lung and colon cancers, respectively, and less frequently in other solid tumors. Recently, the development of covalent KRASG12C inhibitors has shown meaningful anticancer activity in patients. However, many patients with KRASG12C do not respond and/or develop resistance to single-agent treatment. Here we investigate combination therapies that may overcome resistance and broaden patient response to KRASG12C inhibitors. Methods: In vitro preclinical KRASG12C models including lung and colon cancer cell lines were treated with a panel of small molecule and covalent KRASG12C inhibitors. The activity of single-agent versus combination treatments was measured in cell viability assays, and screen hits were validated by in vitro mechanism of action studies. Results: A curated set of 152 compounds was used in combination with screen monitoring cell-viability assays across 12 KRAS mutant lung and colon cancer cell lines. Compounds tested included kinase inhibitors targeting oncogenic signaling pathways, epigenetics modifiers, regulators of apoptosis, chemotherapeutics, and other anticancer agents. The screen identified synergy between covalent KRASG12C and several other inhibitors including those for FGFR, EGFR, and SHP2. Notably, pemigatinib, a potent and selective FGFR1-3 inhibitor, had a significantly high Bliss synergy score. Selectivity and siRNA knockdown experiments were performed to identify the specific FGFR isoform involved in synergistic anticancer activity. Inhibition of FGFR1 activity was shown to be essential, whereas an FGFR2-3 specific inhibitor demonstrated only modest activity in combination with KRASG12C inhibitors. Additionally, FGFR1 knockdown combined with KRASG12C inhibition resulted in increased drug sensitivity; in contrast, knockdown of other FGFR family members did not demonstrate similar increase in sensitivity to KRASG12C inhibition. Conclusions: Cell-viability combination screening identified strong synergy between KRASG12C covalent inhibitors and the FGFR1-3 inhibitor pemigatinib in KRASG12C cancer cell lines. The synergistic effect of pemigatinib and KRASG12C inhibition was notably strongest in mesenchymal cancer cell lines. This effect was further validated in in vitro selectivity and siRNA studies. Together, our results support pemigatinib as a promising agent for combination therapy with KRAS inhibitors in lung cancer. Citation Format: Angela Abdollahi, Margaret Favata, Darlise DiMatteo, Sean Schuette, Erika Boarder, Mark Rupar, Ricardo Macarron, Aidan Gilmartin, Hui Wang, Alejandro Amador-Arjona. Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung 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 412.

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies. Significance: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397

Purpose: Bromodomain and extraterminal domain (BET) proteins regulate the expression of many cancer-associated genes and pathways; BET inhibitors have demonstrated activity in diverse models of hematologic and solid tumors. We report the preclinical characterization of INCB054329, a structurally distinct BET inhibitor that has been investigated in phase I clinical trials. Experimental Design: We used multiple myeloma models to investigate vulnerabilities created by INCB054329 treatment that could inform rational combinations. Results: In addition to c-MYC, INCB054329 decreased expression of oncogenes FGFR3 and NSD2/MMSET/WHSC1, which are deregulated in t(4;14)-rearranged cell lines. The profound suppression of FGFR3 sensitized the t(4;14)-positive cell line OPM-2 to combined treatment with a fibroblast growth factor receptor inhibitor in vivo. In addition, we show that BET inhibition across multiple myeloma cell lines resulted in suppressed interleukin (IL)-6 Janus kinase–signal transducers and activators of transcription (JAK–STAT) signaling. INCB054329 displaced binding of BRD4 to the promoter of IL6 receptor (IL6R) leading to reduced levels of IL6R and diminished signaling through STAT3. Combination with JAK inhibitors (ruxolitinib or itacitinib) further reduced JAK–STAT signaling and synergized to inhibit myeloma cell growth in vitro and in vivo. This combination potentiated tumor growth inhibition in vivo, even in the MM1.S model of myeloma that is not intrinsically sensitive to JAK inhibition alone. Conclusions: Preclinical data reveal insights into vulnerabilities created in myeloma cells by BET protein inhibition and potential strategies that can be leveraged in clinical studies to enhance the activity of INCB054329.

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

BackgroundBlocking the PD-L1 immune checkpoint axis with therapeutic antibodies against either the ligand or PD-1 has proven to be an effective treatment modality for multiple cancer histologies. Small molecules targeting the PD-L1/PD-1 axis represent an alternate modality of blocking this pathway. INCB090244 is a small molecule that blocks the PD-L1/PD-1 interaction and restores T cell function similar to the clinical stage PD-L1 inhibitor INCB086550.MethodsMDA-MB-231 or CHO cells overexpressing PD-L1 were used to investigate effects of INCB090244 on PD-L1 dimerization, and intracellular trafficking. In vivo, CD34+ humanized mice harboring MDA-MB-231 tumors or C57Bl/6 mice bearing GL261 subcutaneous or orthotopic tumors were used to investigate the efficacy, biodistribution, and pharmacodynamic effects of INCB090244. Human specific gene expression changes in tumors from MDA-MB-231 bearing humanized mice were analyzed by RNA sequencing.ResultsIn vitro, INCB090244 potently disrupted the PD-L1:PD-1 interaction, induced PD-L1 dimerization, and inhibited PD-1-mediated negative signaling, resulting in enhanced IFN gamma and IL-2 production in primary human immune cells. Following dimerization, INCB090244 induced internalization of PD-L1 resulting in co-localization with the Golgi apparatus and partial localization in the nucleus. After cell treatment and washing, full restoration of PD-L1 at the cell surface was observed after 5 days of culture in vitro. In vivo, INCB090244 reduced tumor growth in CD34+ humanized mice bearing MDA-MB-231 tumors, to similar levels as atezolizumab. Antitumor activity was completely abrogated in immunodeficient mice, confirming the pharmacologic dependency on a competent immune system. RNA sequencing analysis on tumors from these mice demonstrated similar T cell activation gene signatures as clinical checkpoint blockade antibodies. Biodistribution studies in mice bearing both subcutaneous and orthotopically implanted GL261 glioma tumors demonstrated higher accumulation of INCB090244 in tumor tissue compared to PD-L1 antibodies.ConclusionsINCB090244 effectively disrupted the PD-L1/PD-1 interaction, induced dimerization and internalization of PD-L1, restored immunity in in vitro and in vivo tumor models, and is a suitable surrogate for the clinical candidate INCB086550. RNA sequencing demonstrated T cell activation signatures similar to those observed in patients receiving checkpoint blockade antibodies. Biodistribution studies demonstrated higher subcutaneous and brain tumor penetration by INCB090244 compared to PD-L1 antibodies, suggesting a potential advantage of small molecule PD-L1 inhibitors in accessing intratumoral regions. These data further support the clinical evaluation of small molecule PD-L1 inhibitors as an alternative approach to immune therapy.

Spinal muscular atrophy (SMA), a recessive, neuromuscular disease, is caused by a mutation or deletion in the SMN1 gene. The SMN2 gene is present in the same region of chromosome 5 and is similar in DNA sequence to SMN1 except for a T at position +6 of exon 7 that is likely the predominant functional difference between the two genes. This change alters RNA splicing which results in the removal of exon 7 from the mature mRNA; only 10% full-length transcripts are produced from the SMN2 gene. Our lab has shown that single-stranded oligonucleotides (ODN) can be used to repair genes with single base mutations within the context of the native chromosome. Here, we used SMN2-sequence-specific ODNs to direct the exchange of a T to a C in an SMA skin fibroblast cell line from a type 1 patient. The cells were transfected with ODNs of either 47 or 75 bases in length and designed to hybridize to either the transcribed or non-transcribed DNA strand of the SMN2 gene. We analyzed the genotype of these cells using a well-established Taqman probe-based PCR assay, restriction enzyme digestion, and cycle sequencing. Conversion of the SMN2 genotype to SMN1 was detected when the specific ODN was added. As a result, we observed an increase in production of full-length SMN mRNA, measured by qRT-PCR, and SMN protein, measured by western blotting. Finally, properly localized SMN protein was detected by the accretion of gemini of coiled bodies (gems) only in targeted cells. This is the first report of the use of ODNs to direct genetic conversion of SMN2 to SMN1 in human cells from SMA patients.

Skeletal metastases are often complicated by progression to impending or pathologic fracture and fixation with polymethylmethacrylate (PMMA) bone cement is used for stabilization and pain relief. Adjuvant therapy involving the delivery of PMMA composites mixed with antibiotic or chemotherapeutic agents requires an understanding of the rate of drug diffusion from the cement in addition to measurement of its mechanical properties pre- and postelution of drug. We have developed a method for the analysis of drug diffusion rate and mechanical properties of drug-cement composites using PMMA/methotrexate as a model system. The analysis method revealed the addition of methotrexate to PMMA in concentrations of 1.8 g methotrexate per 40 g PMMA did not change the compression modulus of the cement pre- or postelution of drug. The PMMA/methotrexate composites displayed an average diffusion rate of 50 ng/(mm2)(hour) during the first 6 hours, which decreased to 10 ng/(mm2)(hour) by 36 hours. Diffusion modeling predicts the 20 x 13-mm cylindrical PMMA/methotrexate samples used by the method deliver 10% of the total methotrexate content within 80 hours and 25% of the total within 133 days.

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

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

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

Bromodomains (BD) are protein modules that bind acetylated lysine residues and are components of many epigenetic modifiers and transcription factors. The BET (Bromodomain and extra-terminal) family is composed of four members each harboring two tandem BDs. BET proteins are critical regulators of transcription through interactions with complexes including Mediator and p-TEFb at gene promoter and enhancer elements. Studies using genetic knockdown and small molecule inhibitors have demonstrated that targeting BET proteins is therapeutic in models of cancer and acute inflammation. We describe the preclinical activity of a novel BET inhibitor INCB054329 for the potential treatment of malignant diseases. INCB054329 inhibited binding of BRD2, BRD3 and BRD4 to an acetylated histone H4 peptide with low nanomolar potency. In myeloma cell lines, treatment with INCB054329 inhibited expression of c-MYC and induced HEXIM1. The majority of myeloma, AML, and lymphoma cell lines tested were growth inhibited by INCB054329 with potencies less than 200 nM. Selectivity was seen when compared with nontransformed cells as the potency for growth inhibition of IL-2 stimulated T-cells from normal donors was greater than 1300 nM. Cell cycle analysis revealed treatment-induced G1 arrest. Furthermore in both AML and lymphoma cell lines, INCB054329 induced apoptosis consistent with increased expression of pro-apoptotic regulators. In vivo, oral administration of INCB054329 inhibited tumor growth in several models of hematologic cancers. In the MM1.S multiple myeloma xenograft model, inhibition of tumor growth was correlated with reduction of c-MYC levels. PK-PD analysis showed c-MYC suppression was associated with an IC50 value of less than 100 nM in vivo. In summary these studies demonstrate that INCB054329 is a potent inhibitor of BET transcriptional regulators in models of hematologic malignancies in vitro and in vivo and support its clinical development for the treatment of cancer. Citation Format: Phillip CC Liu, Xuesong Mike Liu, Matthew C. Stubbs, Thomas Maduskuie, Richard Sparks, Nina Zolotarjova, Jun Li, Xiaoming Wen, Margaret Favata, Patricia Feldman, Alla Volgina, Darlise DiMatteo, Robert Collins, Nikoo Falahatpisheh, Padmaja Polam, Yu Li, Maryanne Covington, Sharon Diamond-Fosbenner, Richard Wynn, Timothy Burn, Kris Vaddi, Swamy Yeleswaram, Andrew P. Combs, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Discovery of a novel BET inhibitor INCB054329. [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 3523. doi:10.1158/1538-7445.AM2015-3523

Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. Genomic analyses of squamous cell lung, gastric and urothelial tumors have revealed recurrent genetic alterations in FGFR1, FGFR2 and FGFR3 genes, respectively. FGFR proteins contribute to the development of malignancies by promoting tumor cell proliferation, survival, and migration and supporting angiogenesis. Therefore targeting FGFR kinases may provide therapeutic benefit to patients with cancers that have genetic alterations in genes encoding components of the FGF-FGFR axis. INCB054828 is a potent inhibitor of FGFR1, FGFR2, and FGFR3 that has selective pharmacological activity against cancer cells with FGFR alterations. In vitro, INCB054828 potently inhibited the kinase activity of recombinant FGFR1, FGFR2 and FGFR3 enzymes and was highly selective against a panel of kinases including VEGFR2. In cellular assays, INCB054828 inhibited the autophosphorylation of FGFR proteins with low nanomolar IC50 values and blocked signal transduction by FGFR to downstream markers of pathway activation. Cancer cell lines that have genetic alterations in FGFR1, FGFR2 and FGFR3 were uniquely sensitive to growth inhibition by INCB054828, with IC50 values generally in the range of 3-50 nM, compared with cancer cell lines or normal cells without FGFR dependence (IC50 > 1500 nM). In vivo, once-daily oral administration of INCB054828 inhibited the growth of tumors that are dependent upon FGFR1, FGFR2 and FGFR3 activity at tolerated doses. Suppression of tumor growth was dose-dependent and correlated with pharmacodynamic inhibition of FGFR. Collectively, these preclinical studies demonstrate that INCB054828 potently and selectively inhibits models of FGFR-dependent cancers in vitro and in vivo, supporting the compound's clinical evaluation in patients harboring oncogenic FGFR activation. Citation Format: Phillip CC Liu, Liangxing Wu, Holly Koblish, Kevin Bowman, Yue Zhang, Ronald Klabe, Lynn Leffet, Darlise DiMatteo, Mark Rupar, Karen Gallagher, Michael Hansbury, Colin Zhang, Chunhong He, Paul Collier, Maryanne Covington, Richard Wynn, Swamy Yeleswaram, Kris Vaddi, Timothy Burn, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Preclinical characterization of the selective FGFR inhibitor INCB054828. [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 771. doi:10.1158/1538-7445.AM2015-771

To determine whether maternal age, race, or income, is the primary determinant of breastfeeding continuation rates.Using the Delaware modified-PRAMS 2000 de-identified data set, multivariate modeling of age, income, race, weight, health status, and other breastfeeding continuation factors were analyzed.Breastfeeding continuation at 12 weeks of age was predicted by maternal age independent of race or income. Mothers greater than the mean age were 86% more likely to continue to breastfeed than younger mothers (OR 1.861 95% CI 1.234-2.805). Maternal BMIor = 26, nipple soreness, and breastfeeding difficulty were associated with lower breastfeeding continuation.Maternal age, not race or income, are driving factors in breastfeeding continuation. Physiologic variables, such as maternal obesity, breastfeeding difficulty, and nipple soreness have an adverse influence on duration of breastfeeding. Investment into processes that lengthen breastfeeding duration among mothers, especially younger mothers, who chose to initiate breastfeeding, may have individual and community benefits.

Background Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder that results in the production of a dysfunctional form of the protein, dystrophin. The mdx5cv mouse is a model of DMD in which a point mutation in exon 10 of the dystrophin gene creates an artificial splice site. As a result, a 53 base pair deletion of exon 10 occurs with a coincident creation of a frameshift and a premature stop codon. Using primary myoblasts from mdx5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation. Results Single-stranded DNA oligonucleotides that were designed to repair this splice site mutation corrected the mutation in the gene and restored expression of wild-type dystrophin. This repair was validated at the DNA, RNA and protein level. We also report that the frequency of genetic repair of the mdx mutation can be enhanced if RNAi is used to suppress expression of the recombinase inhibitor protein Msh2 in cultures containing myoblasts but not in those heavily enriched in myoblasts. Conclusion Exogenous manipulations, such as RNAi, are certainly feasible and possibly required to increase the successful application of gene repair in some primary or progenitor muscle cells.