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PDF file - 650K, Figure S1. RSK N-terminal Kinase Directly Phosphorylates YB1 on Ser102. COS7 cells either (A) untransfected or (B) transiently expressing WT RSK2 or T493M RSK2 were placed under serum starvation conditions for 20 hrs, followed by 3 hour treatment with 1microM FMK-PA and subsequently stimulated with 1ng/ml EGF for 10 minutes. The levels of phosphorylated and total RSK2 and YB1 proteins were assessed in total cell lysates using Western Blot. β-actin protein levels were assessed for gel loading control. Figure S2. LJH685 modulates YB1 and BAD phosphorylation in either attached or anchorageindependent setting to similar extent and at similar concentrations of LJH685. MDA-MB-231 cells were treated with the indicated kinase inhibitors for 4 hours. Cell lysates were analyzed by Western blot with indicated antibodies. Figure S3. MDA-MB-231 cells were plated in Matrigel with DMSO or LJH685 for 6 days and colony morphology was examined using phase microscopy. Figure S4. MDA-MB-231 and H358 cells were plated on regular and ultra-low bind tissue culture plates for 5 days in presence of 10 microM LJH685 or DMSO. Cell Cycle distribution was assessed using FACS analysis of propidium iodide stained nuclei.

Supplementary Table S1. Percentage of enzyme activity following dialysis compared to a non-compound treated control; Supplementary Figure S1. PRN1371 inhibited SNU16 cell proliferation as assessed by BrdU incorporation; Supplementary Figure S2. PRN1371 treatment induces apoptosis via caspase activation.

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PDF file - 28K, Percent Inhibition of MDA-MB-231 or H358 cells when grown as colonies on Scivax plates in the presence of LJI308.

The p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases is expressed in a variety of cancers and its substrate phosphorylation has been implicated in direct regulation of cell survival, proliferation, and cell polarity. This study characterizes and presents the most selective and potent RSK inhibitors known to date, LJH685 and LJI308. Structural analysis confirms binding of LJH685 to the RSK2 N-terminal kinase ATP-binding site and reveals that the inhibitor adopts an unusual nonplanar conformation that explains its excellent selectivity for RSK family kinases. LJH685 and LJI308 efficiently inhibit RSK activity in vitro and in cells. Furthermore, cellular inhibition of RSK and its phosphorylation of YB1 on Ser102 correlate closely with inhibition of cell growth, but only in an anchorage-independent growth setting, and in a subset of examined cell lines. Thus, RSK inhibition reveals dynamic functional responses among the inhibitor-sensitive cell lines, underscoring the heterogeneous nature of RSK dependence in cancer.Implications: Two novel potent and selective RSK inhibitors will now allow a full assessment of the potential of RSK as a therapeutic target for oncology. Mol Cancer Res; 12(5); 803–12. ©2014 AACR.

XLSX file - 27K, Competition binding of LJH685, LJI308, BI-D1870-AE, and FMK-PA at 10 microM concentration to either 96 or 492 kinases.

Mutations in MEK1/2 have been described as a resistance mechanism to BRAF/MEK inhibitor treatment. We report the discovery of a novel ATP-competitive MEK1/2 inhibitor with efficacy in wildtype (WT) and mutant MEK12 models. Starting from a HTS hit, we obtained selective, cellularly active compounds that showed equipotent inhibition of WT MEK1/2 and a panel of MEK1/2 mutant cell lines. Using a structure-based approach, the optimization addressed the liabilities by systematic analysis of molecular matched pairs (MMPs) and ligand conformation. Addition of only three heavy atoms to early tool compound

DAY101 (formerly TAK-580) is an oral, CNS-penetrant, selective small molecule pan-RAF kinase inhibitor that is equipotent against BRAF V600E, wildtype BRAF, and CRAF and can inhibit both RAF monomers and dimers, including BRAF fusions. KIAA1549:BRAF fusions have been identified as a key oncogenic driver for pilocytic astrocytoma (Jones et al. 2008). DAY101 has demonstrated significant activity against this fusion preclinically (Sun et al. 2017). Clinical data for DAY101 has shown a durable 63% response rate with good tolerability in children with low-grade glioma (LGG) harboring RAF fusions (Wright et al. 2020). To explore the potential impact of DAY101 in adult solid tumors harboring specific genomic alterations beyond BRAF V600E, a panel of tumor cell lines and PDX models harboring BRAF fusions, non V600 BRAF mutations or RAS mutations were assessed for anti-proliferative activity in response to DAY101. Furthermore, hypothesis-driven MAPK pathway node inhibitor combinations were explored for potential synergy. While performing favourably in preclinical in vivo studies, DAY101 does exhibit some technical challenges in long-term in vitro assays, particularly in those that extend for more than three days. Accordingly, the tool compound TAK-632, which exhibits comparable biochemical and cellular potency to DAY101, was used alternatively as needed for in vitro assays. Single-agent anti-proliferative activity was observed in a melanoma BRAF fusion PDX model treated with TAK-632 ex vivo, with less sensitivity observed in models beyond melanoma harboring other BRAF fusions. Ongoing combination studies with MEK inhibitors demonstrated strong synergy in PDX models harboring BRAF fusions ex vivo, as assessed by Bliss independence or other relevant scoring systems. In cell lines or PDX models harboring non V600 BRAF mutations, in vitro combination studies demonstrated strong synergy when DAY101 was combined with MEK inhibitors, as assessed by Bliss independence score. DAY101 exhibited modest single agent anti-proliferative activity in a panel of KRAS mutant cell lines in vitro and ongoing combination studies will assess potential synergy when combined with MAPK pathway node inhibitors. DAY101 is currently in clinical development for the treatment of patients under 25 years old with LGG harboring RAF alterations, including BRAF fusions and BRAF mutations. Inhibition of both RAF monomers and dimers by DAY101 enables a broader indication selection strategy, beyond LGG and BRAF alterations. Ongoing translational work will highlight the potential utility of DAY101 in adult tumors harboring RAS or RAF alterations, both as a single agent and in hypothesis-driven combinations. Citation Format: Eleni Venetsanakos, Mike Preigh, Jeannie Hou, Michael C. Cox, Jeremy Bender, Sam C. Blackman. DAY101, a potent pan-RAF inhibitor with activity in preclinical models harboring MAPK pathway alterations beyond BRAF V600E mutation [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 1481.

Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clinical validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1-4 for use in oncology indications. An irreversible covalent binding mechanism imparts many desirable pharmacological benefits including high potency, selectivity, and prolonged target inhibition. Herein we report the structure-based design, medicinal chemistry optimization, and unique ADME assays of our irreversible covalent drug discovery program which culminated in the discovery of compound 34 (PRN1371), a highly selective and potent FGFR1-4 inhibitor.

Background: FGFR inhibition is a promising and clinically proven therapeutic approach in a number of solid tumors where genetic alterations of FGFR drive oncogenesis. PRN1371 is a highly selective oral, irreversible inhibitor of FGFR1-4 that exhibits high potency in cancer cell lines harboring FGFR alterations, including mutations and fusions. Methods: Part A of this phase 1 open-label, multicenter clinical trial explored ascending doses of PRN1371 in adult patients with advanced solid tumors who had received at least one prior treatment for recurrent, metastatic, and/or locally advanced disease, and for whom no standard therapy options were anticipated to result in a durable remission. The primary objectives were to investigate the safety and tolerability and determine the maximum tolerated dose (MTD). Secondary objectives included determination of the pharmacokinetic (PK) profile. The trial employed a “3 + 3” design, where cohorts of three patients are studied at each dose level until additional patients need to be added to better assess safety. PRN1371 was dosed once or twice daily in continuous, 28-day cycles until disease progression. Elevated serum phosphorus, a known effect of systemic FGFR inhibition, was managed with oral phosphate-binding medications and a low phosphate diet, with dose interruptions and use of acetazolamide if certain thresholds were exceeded. Results: In part A, PRN1371 was administered to 36 patients in cohorts at doses of 15mg, 20mg, 25mg, and 35mg QD as well as BID doses of 15mg and 25mg. The drug has been generally well tolerated. The most common side effect observed was hyperphosphatemia, which was generally managed by administration of phosphate binders, and there were no treatment-related adverse events leading to study drug discontinuation. Five of 36 patients reported treatment-related serious adverse events (SAEs). PRN1371 was well absorbed, exhibited moderate PK variability with minimal accumulation, and was approximately dose proportional between 15 and 35mg doses. Exposure of parent drug in urine exceeded the cellular IC90 at all dose levels, which may be particularly relevant for localized bladder cancer. Part B of the study has been initiated, which will enroll patients with metastatic urothelial cell carcinoma (mUC) with FGFR1, 2, 3, or 4 genetic alterations. Citation Format: Sarina A. Piha-Paul, Cinta Hierro, Ignacio Matos, Valentina Boni, Noah Hahn, Rhonda Bitting, Todd Bauer, Aggarwal Rahul, Funda Meric-Bernstam, Steven Gourlay, Timothy D. Owens, Ken Brameld, Ann Neale, Richard Schwartz, Steve Murray, Sibel Ucpinar, Peter Foote, Eleni Venetsanakos, Josep Tabernero. A phase 1, multicenter, dose-escalation study of PRN1371, an irreversible covalent FGFR1-4 kinase inhibitor, in patients with advanced solid tumors including metastatic urothelial carcinoma (mUC) [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A19.

An increasing number of cancers are known to harbor mutations, translocations, or amplifications in the fibroblast growth factor receptor (FGFR) family of kinases. The FGFR inhibitors evaluated in clinical trials to date have shown promise at treating these cancers. Here, we describe PRN1371, an irreversible covalent inhibitor of FGFR1-4 targeting a cysteine within the kinase active site. PRN1371 demonstrated strong FGFR potency and excellent kinome-wide selectivity in a number of biochemical and cellular assays, including in various cancer cell lines exhibiting FGFR alterations. Furthermore, PRN1371 maintained FGFR inhibition in vivo, not only when circulating drug levels were high but also after the drug had been cleared from circulation, indicating the possibility of sustained FGFR inhibition in the clinic without the need for continuous drug exposure. Durable tumor regression was also obtained in multiple tumor xenografts and patient-derived tumor xenograft models and was sustained even using an intermittent dosing strategy that provided drug holidays. PRN1371 is currently under clinical investigation for treatment of patients with solid tumors. Mol Cancer Ther; 16(12); 2668–76. ©2017 AACR.

Poly(ADP-ribose) glycohydrolase (PARG) hydrolyzes poly(ADP-ribose) (PAR) chains that are polymerized by PARP enzymes, completing the PAR cycle. Small molecule inhibitors of PARG result in a dose dependent increase in cellular PAR after DNA damage. Here we demonstrate that depletion of XRCC1, a scaffolding protein with an essential role in base-excision repair (BER), sensitizes cancer cells to PARG inhibition. XRCC1 deficient cells exhibit increased nuclear PAR foci in response to PARG inhibition even in the absence of DNA damaging agents. Inhibition of PARP1 with RNAi or small molecule inhibitors rescues cell growth inhibition and reduces the amount of cellular PAR accumulation in PARG inhibitor treated cells. This indicates that the cellular growth inhibition is dependent upon cellular PAR levels, demonstrating selectivity of the small molecule inhibitors for PARG. We hypothesized that inhibition of PAR hydrolysis could result in depletion of cellular NAD as this could prevent recycling of PAR to NAD. Consistent with this hypothesis, PARG inhibition enhanced NAD depletion after treatment of cells with the DNA damaging agent methyl methanesulfonate (MMS). Live cell imaging of XRCC1 depleted cells treated with a PARG inhibitor revealed that cells have large membrane protrusions, similar to the morphology of cells that have been treated with a NAMPT inhibitor, which results in depletion of cellular NAD. Furthermore, addition of the NAD precursor, nicotinamide mononucleotide (NMN) rescued proliferation of PARG inhibited cells. Taken together, these data support a hypothesis in which PARG inhibitors are cytotoxic to sensitive cancer cells via depletion of NAD, ultimately starving the cell of ATP. Thus, PARG inhibition is a novel strategy for exploiting synthetic lethality in cancer cells. The defects that sensitize cancer cells to PARG inhibition are distinct from those that sensitize to PARP inhibitors, namely defects in homology directed repair. Approximately 15% of breast cancer samples exhibit low or no XRCC1 by IHC. A subset (approximately 35%) of the XRCC1 low patient samples also have defects in BRCA1, suggesting that the majority of XRCC1 low tumors may not be responsive to PARP inhibitors. Small molecule PARG inhibitors are currently being evaluated for efficacy in XRCC1 low xenograft models. Citation Format: Leenus Martin, Tzuling Cheng, Dominic I. James, Habiba Begum, Kate M. Smith, Allan Jordan, Ian Waddell, Kedar Vaidya, Marcus Fischer, Bing Yao, Jason Drummond, Leah Cleary, Ruben Martinez, James Sutton, Nandini Ravindran, James Joseph, Eleni Venetsanakos, Michael Dillon, Jeffrey H. Hager, Lisa D. Belmont. PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition [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 1943.

Cancer cells utilize a variety of pathways to generate an immune permissive growth environment and evade the host anti-tumor immune response. One such pathway is production of T-cell inhibitory tryptophan (Trp) metabolites, kynurenine and kynurenic acid (KYN), via up-regulation of indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO2) and up-regulation of the KYN- receptor (aryl hydrocarbon receptor, AhR) in infiltrating lymphoid and myeloid cells. Through AhR activation, KYN both directly and indirectly, via stimulation of regulatory T-cells (Tregs), dendritic cells (DCs) and tumor-associated macrophages (TAM), modulates CD8+ and CD4+ T-cell function resulting in potent inhibition of anti-tumor immunity. Inhibition of AhR activation therefore represents a novel approach to blocking the immuno-inhibitory effects of Trp metabolites generated by endogenous IDO1 and TDO2. To thwart the effects of KYN and other immunosuppressive AhR agonist ligands, we have generated a series of small-molecule antagonists of the human and mouse AhR. To further analyze the effects of AhR inhibition on immuno-modulation and tumor growth, we selected from our series a highly potent antagonist of AhR (IDB-AHRi). In an XRE-dependent luciferase reporter system, IDB-AHRi blocked activation of AhR by 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) with an IC50 of 111 and 3 nM in human HEPG2 and mouse HEPA1C1C7 cell lines respectively. IDB-AHRi inhibited translocation of cytoplasmic AhR to the nucleus and protected AhR from TCDD-induced degradation. When human peripheral blood mononuclear cells were treated with IDB-AHRi ex-vivo, expression of IFN-γ and TNF-α was significantly enhanced, an effect that could be reversed with the addition of TCDD. In vivo, IDB-AHRi dosed at 30 mg/kg (PO QD) in the CT26 colon carcinoma syngeneic mouse model resulted in a reduction of tumor growth, an increase in tumor-infiltrated CD45+CD8+Ki67high T-cells and a significant reduction in the frequency of tumor infiltrated FoxP3+CD4+ regulatory T-cells and tumor associated M2-like macrophages. In summary, this data demonstrates that blocking AhR with a highly potent antagonist can significantly enhance T-cell activity in vitro and drive anti-tumor immunity in vivo, resulting in reduced tumor growth. Citation Format: James Joseph, Marcos Gonzalez-Lopez, Christina Galang, Candy Garcia, Hadia Lemar, Jing Lu, Kedar Vaidya, Marcus Fischer, Christian Frey, Muzaffar Alam, Bing Yao, Michael Dillon, Jeffrey H. Hager, Eleni Venetsanakos, Fred Aswad. Small-molecule antagonists of the Aryl Hydrocarbon Receptor (AhR) promote activation of human PBMCs in vitro and demonstrate significant impact on tumor growth and immune modulation in vivo [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 4719.

Introduction: Multiple human cancers harbor alterations in FGFRs that drive tumor growth, including mutations, translocations and amplifications. PRN1371 is an irreversible, covalent FGFR1-4 inhibitor that exhibits highly selective and sustained inhibition of FGFR which extends well beyond circulating drug concentrations in preclinical models. The duration of inhibition of the FGFR signaling pathway is dependent on protein turnover of FGFR, which may vary depending on the type of FGFR alteration. Thus, we set out to investigate whether the duration of target inhibition differs across cancer cell lines of various lineages harboring different FGFR alterations, including fusions and mutations. Furthermore, as FGFR inhibitors exhibit hyperphosphatemia via on-target pharmacology, we also investigated the duration of target inhibition in primary renal epithelial cells which are wild-type for FGFR. Materials and Methods: Cancer cell lines from several lineages harboring different FGFR alterations were treated with increasing concentrations of PRN1371 in vitro for 1 hour, before washing out the compound. Cells were harvested at various time-points post-washout, protein lysates were generated and assessed for modulation of the downstream signaling pathway by western blot analysis. Results: Dose-dependent inhibition of phospho-ERK was observed in the cancer cell lines tested in response to compound treatment for 1 hour in vitro. Dose dependent partial or full rebound of phospho-ERK back to baseline levels were detected in cancer cell lines after a prolonged period post-washout. In contrast, full rebound of phospho-ERK was observed at 1 hour post washout in response to a non-covalent inhibitor. Studies are on-going to assess duration of pathway inhibition in the primary renal proximal epithelial cells. Conclusions: PRN1371 is a potent, highly selective irreversible inhibitor exhibiting sustained inhibition of FGFR signaling across cancer cell lines harboring different FGFR alterations. The duration of inhibition of the downstream signaling was comparable across cancer cell lines harboring different FGFR alterations, including mutations, fusions and amplification of FGFR and was prolonged when compared to a non-covalent inhibitor. A Phase 1 clinical trial of PRN1371 for the treatment of solid tumors harboring FGFR alterations is ongoing (NCT02608125). Citation Format: Eleni Venetsanakos, Yan Xing, Natalie Loewenstein, J. Michael Bradshaw, Dane Karr, Jacob LaStant, Philip Nunn, Jin Shu, Abha Bommireddi, Jens Oliver Funk, David M. Goldstein, Stefani Wolff, Ken A. Brameld, Steven G. Gourlay. PRN1371, an irreversible, covalent inhibitor of FGFR1-4 exhibits sustained pathway inhibition in cancer cell lines [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 2091. doi:10.1158/1538-7445.AM2017-2091

TPS2616 Background: FGFR inhibition is a promising and clinically proven therapeutic approach in a number of solid tumors where genetic alterations of FGFR drive oncogenesis. PRN1371 is a highly selective oral, irreversible inhibitor of FGFR1-4 that exhibits high potency in cancer cell lines harboring FGFR alterations, including mutations and fusions. Methods: Part A of this phase 1 clinical trial explores ascending doses of PRN1371 in adult patients with advanced solid tumors in a "3 + 3" design, where cohorts of three patients are studied at each level until additional patients need to be added to better assess safety, establish the maximum tolerated dose and define the recommended phase 2 dose (RP2D). PRN1371 is dosed once or twice daily in continuous, 28-day cycles until disease progression. Part B studies include two or three expansion cohorts of different tumor types, 10 patients each with FGFR1-4 gene mutations, fusions, or amplification at the RP2D. The on-target effect of serum phosphorus and FGF23 increases are measured as potential pharmacodynamic biomarkers. Elevated serum phosphorus is managed with oral phosphate binding medications and a low phosphate diet, with dose interruptions and use of acetazolamide if certain thresholds are exceeded. Circulating tumor DNA from patients at baseline and during follow up is analyzed for FGFR genetic alterations. Pre and on-treatment tumor biopsies in Part B will be tested for a panel of pharmacodynamic biomarkers of FGFR inhibition. Clinical trial information: NCT02608125.

The p90 ribosomal S6 kinase (RSK) family of serine/threonine kinases is expressed in a variety of cancers and its substrate phosphorylation has been implicated in direct regulation of cell survival, proliferation, and cell polarity. This study characterizes and presents the most selective and potent RSK inhibitors known to date, LJH685 and LJI308. Structural analysis confirms binding of LJH685 to the RSK2 N-terminal kinase ATP-binding site and reveals that the inhibitor adopts an unusual nonplanar conformation that explains its excellent selectivity for RSK family kinases. LJH685 and LJI308 efficiently inhibit RSK activity in vitro and in cells. Furthermore, cellular inhibition of RSK and its phosphorylation of YB1 on Ser102 correlate closely with inhibition of cell growth, but only in an anchorage-independent growth setting, and in a subset of examined cell lines. Thus, RSK inhibition reveals dynamic functional responses among the inhibitor-sensitive cell lines, underscoring the heterogeneous nature of RSK dependence in cancer. Implications: Two novel potent and selective RSK inhibitors will now allow a full assessment of the potential of RSK as a therapeutic target for oncology. Mol Cancer Res; 12(5); 803–12. ©2014 AACR.

Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf-MEK-extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of alpha6- and beta3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface alpha6- and beta3-integrin expression. Induced beta3-integrin was expressed on the cell surface as a heterodimer with alphav-integrin; however, the overall level of alphav-integrin expression was not altered by Ras or Raf. Raf-induced beta3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce beta3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated beta3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, beta3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on beta3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface alphavbeta3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

Malignant mesothelioma (MM) is a fatal solid tumor of the mesothelium for which there is currently no ameliorating treatment. Using our murine model of this malignancy, which closely resembles the human disease, we have shown that immunotherapy may be of value in the treatment of MM. Because recombinant interleukin-12 (rIL-12) has strong immunomodulatory effects in vivo, we studied the effects of rIL-12 on murine antitumor immune responses, using a nonimmunogenic murine MM tumor cell line (AB1) in vivo. Systemic administration of rIL-12 at the time of tumor inoculation prevented AB1 tumor growth in up to 70% of treated mice, 50% of which were still resistant to AB1 upon rechallenge, indicating that long-term immunologic antitumor effects had been established. This rIL-12-induced effect was dependent on the involvement of both CD4(+) and CD8(+) but not natural killer (NK) cells. Importantly, treatment of established tumors with intralesional injections of rIL-12 resulted in temporary tumor regression or growth inhibition. This effect was dependent on the continuous presence of rIL-12 and correlated with increased numbers of CD4(+) and CD8(+) cells infiltrating the remaining tumor mass. Effective inhibition of tumor growth also occurred when IL-12 was released within MM tumors by coadministration of MM cells that had been stably transfected with the gene for IL-12. These data indicate that IL-12 has potential in the immunotherapy of MM, through gene transfer or local cytokine administration, provided that significant intratumor levels of IL-12 can be achieved for prolonged periods.

Introduction: Multiple human cancers harbor alterations in FGFRs that drive tumor growth, including mutations, translocations and amplifications. We developed a covalent, irreversible, highly selective FGFR1, 2, 3 and 4 inhibitor, PRN1371, by targeting a cysteine residue within the kinase domain. This approach enables highly selective and sustained inhibition of FGFR which extends well beyond circulating drug concentrations. PRN1371 is currently in a phase 1 clinical trial for the treatment of solid tumors. Materials and Methods: FGFR1-4 enzymatic activities were measured using the Caliper microfluidics Labchip system. Inhibition of proliferation was assessed over 3 days using Cell-Titer-Glo. SNU16 and RT4 xenograft tumor models were used to investigate pharmacodynamics and efficacy. For tumor inoculation, cancer cells were implanted into the rear flank of immunocompromised mice. Once tumor volume reached a mean average of 175mm3, mice were randomized and treated with FGFR inhibitor. Results: PRN1371 is a novel investigative drug that is a potent ATP competitive, highly selective inhibitor of the FGFR family of protein kinases. PRN1371 binds irreversibly to FGFR1, 2, 3 and 4, leading to long duration of target inhibition. The IC50 values for FGFR1, 2, 3 and 4 are 0.7, 1.3, 4.1 and 19.2 nM, respectively. The IC50 values are 55.0, 12.0, 1.2 and 2.3 nM for FGFR3-V555M, FGFR2-N549H, FGFR3-G697C and FGFR3-K650E, respectively. PRN1371 exhibited no significant activity against 247 other protein kinases. PRN1371 demonstrates potent inhibition of cell proliferation in multiple tumor cell lines driven by dysregulated FGFR signaling. In RT4, RT112, SNU16, AN3-CA, LI7, JHH7, and OPM2 cells, EC50's are 4.0, 4.1, 2.6, 43.3, 33.1, 231 and 14.0 nM, respectively. PRN1371 is rapidly and well-absorbed. Bioavailability is dose and species-dependent. PRN1371 demonstrates potent tumor growth inhibition and regression in FGFR3-fusion expressing RT4 and FGFR2-amplified SNU16 xenograft models, when dosed either continuously, or intermittently, and is generally well-tolerated. PRN1371 is also shown to have significant anti-tumor activity against a panel of PDX tumor xenografts of various lineages harboring different FGFR pathway alterations. The phase 1 clinical trial will evaluate ascending doses of PRN1371 in a conventional “three plus three” design. Expansion cohorts of selected tumor types will then be investigated at the recommended dose level. Conclusion: PRN1371 is a potent, highly selective, irreversible inhibitor exhibiting sustained inhibition of FGFR 1, 2, 3 and 4. A Phase 1 clinical trial of PRN1371 for the treatment of solid tumors is currently ongoing. Citation Format: Eleni Venetsanakos, Michael Bradshaw, David M. Goldstein, Kwan Leung, Dane Karr, Yan Xing, Jacob LaStant, Philip Nunn, Jin Shu, Abha Bommireddi, Steven G. Gourlay, Jens Oliver Funk, Ken A. Brameld. PRN1371, an irreversible, covalent inhibitor of FGFR1, 2, 3 and 4 is highly efficacious in preclinical tumor models. [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 1249.

TPS2602Background: FGFR inhibition is a promising therapeutic approach in a number of solid tumors where genetic alterations of FGFR drive tumor cell proliferation, and survival. PRN1371 is an oral...

To facilitate the study of human endothelial cells we have used a replication defective retrovirus encoding the catalytic subunit of telomerase (hTERT) to derive populations of telomerase-immortalized human microvascular endothelial (TIME) cells. Whereas parental HMVECs became senescent on average within 35-45 population doublings (PDs), TIME cells have continued to proliferate for at least 200 PDs. TIME cells express readily detectable telomerase activity but display only a modest increase in telomere length. Karyotypic analysis reveals the cells to have a normal complement of human chromosomes with no evidence of gross genetic abnormalities. Furthermore, TIME cells retain many of the characteristics of the primary endothelial cells from which they were derived. For example, they express a panel of characteristic endothelial cell surface marker proteins such as CD31/PECAM-1 and alpha(v)beta3-integrin. In addition, TIME cells express receptors for low-density lipoprotein (LDL) receptor as they are competent for receptor-mediated endocytosis of fluorescent acetylated LDL. Importantly, when plated on matrigel, TIME cells undergo tubule formation. Moreover, when cocultured in the presence of human glioma cells, but not primary human astrocytes, TIME cells are induced to form stable tubules. Detachment of TIME cells from extracellular matrix leads to a form of programmed cell death known as anoikis. Conditional activation of the protein kinase Akt (Akt:ER*) significantly inhibited the onset of TIME cell anoikis under these conditions. We believe that the ability of hTERT to immortalize primary human endothelial cells, and the fact that such cells retain the endothelial characteristics of the cells from which they were derived, will greatly facilitate the analysis of human endothelial cell biology in vitro.

Malignant mesothelioma (MM) is a solid tumor of the mesothelium for which there is no curative treatment. MM appears to be sensitive to immunotherapeutic approaches, and one of the most powerful immunomodulatory cytokines with antitumor effects is interleukin (IL)-12. We have previously shown in a murine model of MM that systemic administration of recombinant IL-12 induces a potent anti-MM immune response. The nature and accessibility of MM tumors means that they are suitable candidates for direct cytokine and gene-transfer therapeutic approaches. Therefore, we undertook a study to assess the antitumor effects induced by the local production of IL-12 within MM tumors by transfecting a murine MM line with the genes for IL-12. The IL-12 transfectant (AB1-IL-12) did not produce tumors in normal mice, but did so in athymic nude mice, implicating T cells in the prevention of MM tumor growth. In mixing experiments, paracrine IL-12 production inhibited growth of untransfected MM cells provided that cells producing IL-12 represented more than 50-80% of the inoculum. Furthermore, BALB/c mice previously challenged with AB1-IL-12 were protected against rechallenge with parental AB1 tumor, indicating that the transfectant induced long-term immunity. AB1-IL-12 induced systemic immunity that was effective at reducing the incidence of parental AB1 tumor at a distal site, but its effects were dose-dependent. Though both CD4(+) and CD8(+) cells infiltrated the rejecting tumor, CD8(+) effector cells were essential for protection against development of parental AB1 tumor. This study shows that paracrine secretion of IL-12, generated by gene transfer, can induce immunity against MM that can act locally and also at a distant site. In addition, there was no evidence of toxicity, which has been associated with the systemic administration of IL-12, indicating that this cytokine is a good candidate for experimental gene therapy in MM.