Activity In Multiple Preclinical Models Research Articles

Inhibition of CDK1 by RO-3306 Exhibits Anti-Tumorigenic Effects in Ovarian Cancer Cells and a Transgenic Mouse Model of Ovarian Cancer.

Ovarian cancer is the deadliest gynecological malignancy of the reproductive organs in the United States. Cyclin-dependent kinase 1 (CDK1) is an important cell cycle regulatory protein that specifically controls the G2/M phase transition of the cell cycle. RO-3306 is a selective, ATP-competitive, and cell-permeable CDK1 inhibitor that shows potent anti-tumor activity in multiple pre-clinical models. In this study, we investigated the effect of CDK1 expression on the prognosis of patients with ovarian cancer and the anti-tumorigenic effect of RO-3306 in both ovarian cancer cell lines and a genetically engineered mouse model of high-grade serous ovarian cancer (KpB model). In 147 patients with epithelial ovarian cancer, the overexpression of CDK1 was significantly associated with poor prognosis compared with a low expression group. RO-3306 significantly inhibited cellular proliferation, induced apoptosis, caused cellular stress, and reduced cell migration. The treatment of KpB mice with RO-3306 for four weeks showed a significant decrease in tumor weight under obese and lean conditions without obvious side effects. Overall, our results demonstrate that the inhibition of CDK1 activity by RO-3306 effectively reduces cell proliferation and tumor growth, providing biological evidence for future clinical trials of CDK1 inhibitors in ovarian cancer.

Abstract 5294: MDX-124, a novel annexin-A1 antibody, induces significant anti-cancer activity in multiple preclinical models

Abstract Annexin-A1 (ANXA1) is secreted from both cancer and immune cells in response to several physiological stimuli and modulates cellular functions through interactions with formyl peptide receptors (FPR1/2). Overexpression of ANXA1 has been observed in multiple cancers, including triple-negative breast (TNBC), colorectal, lung, pancreatic, gastric and prostate, and correlates with poor prognosis and decreased overall survival. ANXA1 has also been shown to promote cancer cell proliferation, angiogenesis, migration and drug resistance, and to modulate the tumor microenvironment. MDX-124 is a novel humanized antibody targeting ANXA1. Previously we presented data demonstrating its significant anti-proliferative activity. Here we provide further data on the mechanism of action of MDX-124, notably its impact on tumor growth, cell cycle arrest and migration in several preclinical cancer models. Incubation of pancreatic (BxPC-3), lung (A549) and TNBC (MDA-MB-231) cancer cell lines with MDX-124 for 24 h decreased the proportion of cells in S-phase by up to 18.3% with a concomitant increase in G1 phase of up to 33.5% versus untreated cells. This effect occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest. After 72 h incubation with MDX-124, the migratory ability of gastric (AGS), prostate (PC-3), TNBC (MDA-MB-231), lung (A549), pancreatic (MIA PaCa-2) and colorectal (LoVo) cancer cells was significantly reduced in a dose-dependent manner when compared to untreated controls. Proteomic analysis following incubation of MDX-124 with a panel of cancer cell lines for 72 h demonstrated substantial alterations in the level of expression and phosphorylation of multiple key oncogenic proteins. In the MycCaP-Bo syngeneic model of bone metastatic prostate cancer, mice treated with the murine analog of MDX-124 (10 mg/kg, BIW) had a 52% reduction in mean tumor growth after 14 days compared to isotype control treated mice. In conclusion, our data indicate that targeting ANXA1 with MDX-124 inhibits key tumorigenic processes in several clinically challenging cancer indications. MDX-124 therefore provides an innovative approach to cancer therapy. Medannex initiated a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies known to overexpress ANXA1. Citation Format: Fiona C. Dempsey, Hussein Al-Ali, Scott J. Crichton, Charlene Fabian, Chris Pepper, Bin-Zhi Qian, Xue-Feng Li, Christopher N. Parris. MDX-124, a novel annexin-A1 antibody, induces significant anti-cancer activity in multiple preclinical 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 5294.

Abstract 1874: MDX-124, a novel annexin-A1 antibody, induces an anti-tumor immune response and wide-ranging anti-cancer activity in multiple preclinical models

Abstract Major recent advances have increased the understanding of both the molecular mechanisms and the immune response in cancer. This has led to the discovery of new agents that regulate the tumor immune microenvironment. Annexin-A1 (ANXA1) is a phospholipid binding protein that has been shown to have immunomodulatory effects on T-cells, macrophages and dendritic cells. Although normally localized in the cytoplasm, ANXA1 is secreted in response to a number of different stimuli and can modulate cellular functions through interactions with formyl peptide receptors (FPR1/2). Overexpression of ANXA1 has been observed in several cancer types including triple-negative breast (TNBC), lung, pancreatic and ovarian, and correlates with poor prognosis and decreased overall survival. Furthermore, ANXA1 has also been shown to influence cancer cell proliferation, angiogenesis, migration and drug resistance. Hence, we explored the role of MDX-124, a novel humanized antibody targeting ANXA1, and here present data from several pre-clinical cancer models. Results: Incubation of a panel of cell lines representing several histological cancer subtypes with MDX-124 for 72h resulted in a statistically significant dose-dependent reduction in cell viability compared to control. ANXA1 expression in the subcellular compartments (nucleus, cytoplasm and membrane) was quantified in a panel of cancer cell lines via imaging flow cytometry and the anti-proliferative effect of MDX-124 was shown to correlate with membrane ANXA1 expression. Using a transwell assay, TNBC and acute myeloid leukemia cell lines exposed to MDX-124 exhibited a statistically significant reduction in migration compared to untreated controls. We also observed that MDX-124 induced antibody dependent cellular cytotoxicity (ADCC) activity in a dose-dependent manner using a reporter bioassay (EC50 = 0.38 nM). MDX-124 was further evaluated in an additional panel of in-vitro assays and shown to exert consistent anti-cancer activity. In conclusion, our data indicate that targeting ANXA1 with MDX-124 inhibits tumorigenic processes and induces an immune response in tumors overexpressing ANXA1. MDX-124 therefore provides an innovative approach to cancer therapy. Investigations are continuing to explore the efficacy of MDX-124 in syngeneic mouse models as both a single agent and in combination with standard of care agents. Citation Format: Fiona C. Dempsey, Hussein Al-Ali, Scott J. Crichton, Chris Pepper, Christopher N. Parris. MDX-124, a novel annexin-A1 antibody, induces an anti-tumor immune response and wide-ranging anti-cancer activity in multiple preclinical models [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 1874.

Targeting the oncogene LSF with either the small molecule inhibitor FQI1 or siRNA causes mitotic delays with unaligned chromosomes, resulting in cell death or senescence

BackgroundThe oncogene LSF (encoded by TFCP2) has been proposed as a novel therapeutic target for multiple cancers. LSF overexpression in patient tumors correlates with poor prognosis in particular for both hepatocellular carcinoma and colorectal cancer. The limited treatment outcomes for these diseases and disappointing clinical results, in particular, for hepatocellular carcinoma in molecularly targeted therapies targeting cellular receptors and kinases, underscore the need for molecularly targeting novel mechanisms. LSF small molecule inhibitors, Factor Quinolinone Inhibitors (FQIs), have exhibited robust anti-tumor activity in multiple pre-clinical models, with no observable toxicity.MethodsTo understand how the LSF inhibitors impact cancer cell proliferation, we characterized the cellular phenotypes that result from loss of LSF activity. Cell proliferation and cell cycle progression were analyzed, using HeLa cells as a model cancer cell line responsive to FQI1. Cell cycle progression was studied either by time lapse microscopy or by bulk synchronization of cell populations to ensure accuracy in interpretation of the outcomes. In order to test for biological specificity of targeting LSF by FQI1, results were compared after treatment with either FQI1 or siRNA targeting LSF.ResultsHighly similar cellular phenotypes are observed upon treatments with FQI1 and siRNA targeting LSF. Along with similar effects on two cellular biomarkers, inhibition of LSF activity by either mechanism induced a strong delay or arrest prior to metaphase as cells progressed through mitosis, with condensed, but unaligned, chromosomes. This mitotic disruption in both cases resulted in improper cellular division leading to multiple outcomes: multi-nucleation, apoptosis, and cellular senescence.ConclusionsThese data strongly support that cellular phenotypes observed upon FQI1 treatment are due specifically to the loss of LSF activity. Specific inhibition of LSF by either small molecules or siRNA results in severe mitotic defects, leading to cell death or senescence - consequences that are desirable in combating cancer. Taken together, these findings confirm that LSF is a promising target for cancer treatment. Furthermore, this study provides further support for developing FQIs or other LSF inhibitory strategies as treatment for LSF-related cancers with high unmet medical needs.

Abstract C14: A novel RAD51 inhibitor, CYT-0851, shows anticancer activity in preclinical models of pancreatic cancer

Abstract Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. We leverage a “gain-of-function” synthetic lethality approach to selectively target cancers that ectopically express Activation Induced Cytidine Deaminase (AID). AID is a DNA-directed cytidine deaminase normally transiently expressed in maturing B-lymphocytes where it plays a critical role in somatic hypermutation and immunoglobulin class switching. However, AID is dysregulated and inordinately expressed in many cancers, where it acts as a DNA-damaging enzyme mutating widespread locations throughout the genome. This activity leads to the accumulation of point mutations and DNA breaks and promotes DNA replication stress. In the latter context, AID-expressing cells become critically dependent on the homologous recombination factor RAD51 to survive DNA damage-induced replication stress. We have developed a novel small-molecule inhibitor of RAD51, CYT-0851, that is preferentially cytotoxic to AID expressing cells. We have shown in vitro and in vivo that CYT-0851 is efficacious in AID-expressing lymphoma and leukemia mouse models. Genome profiling and gene expression studies have shown many solid tumors, including pancreatic cancer, also aberrantly express AID. To determine whether targeting RAD51 may be an effective approach to the treatment of AID-expressing pancreatic cancer, we evaluated the antitumor activity of CYT-0851 in multiple patient-derived pancreatic cancer xenografts (PDX) models with a range of AID expression levels. All models tested showed tumor growth inhibition (TGI), ranging from 63 to 104% with CYT-0851 treatment. A crossover experiment was performed with one model to determine the activity of CYT-0851 in large, established tumors. We observed tumor regression (137% TGI) in large tumors upon treatment with CYT-0851, resulting in one partial responder and one tumor-free responder. The data presented here demonstrate that CYT-0851 exerts anticancer activity in multiple preclinical models of pancreatic cancer. Considering that recent published studies have shown that these cancers can ectopically overexpress AID, we conclude that CYT-0851 may be an effective treatment for pancreatic cancer. Overall, this provides a rationale for the continued development of CYT-0851 as a novel therapeutic for pancreatic cancer patients. Citation Format: Melinda Day, Jean-Marc Lapierre, Tom O'Shea, Kevin Mills. A novel RAD51 inhibitor, CYT-0851, shows anticancer activity in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C14.

Abstract 2087: Mechanistic studies reveal novel molecular insights on anticancer properties of F10 compound in human colon cancer cells

Abstract Fluoropyrimidine (FP) drugs, mainly 5-Fluorouracil (5-FU), constitute the backbone of combination chemotherapy regimens (e.g. FOLFOX and FOLFIRI) for treating colorectal cancer (CRC) and provide a survival benefit for patients with stage II, III, and IV colorectal cancer. However, 5 year survival rate for CRC patients with metastatic disease is less than 10%. This dreadful situation suggests urgent need for novel and more potent FPs to improve the outcomes of these patients. We have developed novel fluoropyrimidine drug polymers (e.g. F10) that show promising antitumor activity in multiple pre-clinical models even while displaying reduced systemic toxicities. F10 is taken up by malignant cells via active transport compared to 5-Fluorouracil, which is taken up by diffusion in malignant and non-malignant cells. Additionally, F10 compound is primarily incorporated into DNA, and produced thymidylate synthase and topoisomerase toxic metabolites at greater levels, whereas 5-Fluorouracil is primarily incorporated into RNA, and only has about 5-10% production of active form of thymidylate synthase toxic metabolites. MMR is involved in repairing the DNA mismatches and FP drugs induced DNA damage. Interestingly, our studies show that F10 acts by different mechanisms compared to 5-FU. Particularly, F10 treated CRC cells exhibit replication stress as evidenced by paused and slow progression of replication forks and R-loops formation at the active transcriptional regions, suggesting replication and transcription dependent generation of DNA lesions in these cells. F10 treatment induced several fold increased DNA double strand breaks (DSB) as measured by COMET assays and about 1000 folds more cytotoxicity to CRC cells compared to 5-FU. Similarly, F10 treatment activated distinctive DNA damage responses in MMR proficient CRC cells compared MMR deficient cells. Additionally, our studies suggest F10 as a promising therapeutic agent for CRC therapy either alone or in combination with CHK1 and PARP inhibitors. Citation Format: Chinnadurai Mani, William Gmeiner, Komaraiah Palle. Mechanistic studies reveal novel molecular insights on anticancer properties of F10 compound in human colon cancer cells [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 2087.

TP-0184 Inhibits ALK2/ACVR1, Decreases Hepcidin Levels, and Demonstrates Activity in Preclinical Mouse Models of Functional Iron Deficiency

BackgroundHepcidin is a liver peptide hormone that functions as a master regulator of bioavailable iron. Hepcidin levels become constitutively high in chronic inflammatory conditions where functional iron deficiency (FID) becomes a challenge. Approaches to target hepcidin expression or function have emerged as attractive strategies to reverse the complications of functional iron deficiency. Targeting the activation of the ALK2/ACVR1 receptor has been validated as a promising approach to target hepcidin. Activation of ALK2/ACVR1 in the liver induces the SMAD-driven transcription of hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Current therapeutic approaches for anemia of chronic disease (ACD) rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which adequately address the underlying pathological deficit of functionally low iron levels. TP-0184 has been developed as a potent and selective inhibitor of ALK2/ACVR1, and it was hypothesized that targeting of ALK2/ACVR1 with TP-0184 would reverse the effects of FID in preclinical models of anemia. TP-0184 is a small-molecule inhibitor of ALK2 kinase activity, exhibiting an IC50 of 5 nM in biochemical assays.AimsThe current study seeks to establish proof-of-concept that TP-0184 targets ALK2/ACVR1 and reverses the effects of FID. Utilizing the unique experience in a single patient IND, the clinical effects of TP-0184 on serum hepcidin levels was evaluated.MethodsMultiple preclinical models of anemia were used to evaluate the efficacy of TP-0184. These models included: a short-term chemical induced anemia by turpentine oil (TO), a model of anemia of cancer using the TC-1 syngenic lung cancer cell line, and an infectious disease model using heat-inactivated Brucella abortus (HKBA).ResultsTP-0184 has demonstrated consistent preclinical activity in three mouse efficacy models of FID. In the first model, TP-0184 reversed hepcidin induction more than 6.8-fold in mice induced with TO. The simplicity of the TO model has allowed the exploration of numerous dose levels and treatment schedules of TP-0184 and the ability to correlate these with pharmacokinetic plasma levels of the compound. In the second model, TP-0184 reversed elevated hepcidin levels and increased iron levels induced in TC-1 tumor bearing mice. Hepcidin was reduced nearly 3-fold with doses as low as 25 mg/kg. In the third model, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection of HKBA. In addition to these preclinical models, a single cancer patient received oral administration of TP-0184 in an emergency setting. Although the primary objectives of the single patient study did not include anemia, pharmacokinetic and pharmacodynamic measurements related to anemia were measured in the patient. The patient was administered 2 cycles of TP-0184, where each cycle evaluated a different formulation. Before the first cycle, the patient had a baseline serum hepcidin level of 559 pg/mL. These levels dropped to below the detectable limit within 24 hours of the first dose with TP-0184. Hepcidin levels remained undetectable for the duration of TP-0184 treatment. After a break in treatment, the patient was administered a second cycle of TP-0184 in an alternative formulation. During this cycle, the patient's hepcidin levels decreased from 182 pg/mL to 69 pg/mL after 24 hours of the first dose (cycle 2). With daily dosing, the patient's serum hepcidin levels continued to decrease to undetectable levels by at least 9 days. Importantly, serum iron levels mirrored serum hepcidin levels well. Plasma pharmacokinetics were also obtained in this patient.ConclusionTargeting ALK2/ACVR1 is an attractive strategy to downregulate hepcidin levels and reverse the effects of FID. TP-0184, a potent ALK2/ACVR1 inhibitor, has demonstrated consistent activity in multiple preclinical models and now has preliminary proof-of-concept evidence that it can modulate hepcidin levels in man. The development of TP-0184 is currently progressing through preclinical development and a Phase I first-in-human study focusing on FID is currently being planned. DisclosuresPeterson:Tolero Pharmaceuticals, Inc.: Employment. K:Tolero Pharmaceuticals, Inc.: Employment. Whatcott:Tolero Pharmaceuticals, Inc.: Employment. Siddiqui-Jain:Tolero Pharmaceuticals, Inc.: Employment. Weitman:Tolero Pharmaceuticals, Inc.: Employment. Bearss:Tolero Pharmaceuticals, Inc.: Employment. Warner:Tolero Pharmaceuticals, Inc.: Employment.

Abstract 1151: SY-1365, a potent and selective CDK7 inhibitor, exhibits promising anti-tumor activity in multiple preclinical models of aggressive solid tumors

Abstract CDK7 has recently emerged as an attractive target in cancer since its inhibition decreases the transcript levels of oncogenic transcription factors, especially those driven by super-enhancers (SEs). Cancers have been hypothesized to be addicted to SE regulated genes and simultaneous suppression of multiple SE associated genes through CDK7 inhibition might represent a novel, powerful way to selectively kill cancer cells. Previously, we reported that SY-1365, a highly selective covalent CDK7 inhibitor, induces apoptosis in leukemia cells, but not in non-malignant cells, and demonstrates anti-tumor activity in in vivo models of leukemia. In this study, we extend these findings to the identification of multiple solid tumors that are susceptible to SY-1365 and compare the effects of SY-1365 on gene expression to other gene control agents. SY-1365 was screened in a panel of solid tumor cell lines, revealing activity in breast, ovarian, colorectal and lung cancer cells with low nM EC50 and rapid induction of apoptosis. In breast cancer, a subset of triple negative breast cancer (TNBC) cell lines were found to be more sensitive than luminal breast cancer cell lines, so we extended our studies to in vivo models and showed substantial tumor growth inhibition in multiple patient-derived xenograft (PDX) models of TNBC. Since other compounds have been reported to modulate the expression of SE regulated genes, we compared the transcriptional effects of SY-1365 treatment with those of a pan-CDK inhibitor (flavopiridol), a CDK9 inhibitor (NVP2) and a BRD4 inhibitor (JQ1) using microarray analysis. Applying principal component analysis, we observed a unique transcriptional response elicited by SY-1365 compared to the other inhibitors. NVP2 and flavopiridol inhibited an overlapping and much larger gene set than either JQ1 or SY-1365. Interestingly, SY-1365 treatment decreased the expression of oncogenic transcription factors, cell cycle checkpoint regulators and DNA damage response genes. The downregulation of transcripts involved in apoptosis and DNA damage response by SY-1365 suggests that CDK7 inhibition might synergize with targeted agents that affect these processes. Indeed, we observed that SY-1365 was synergistic with the PARP inhibitor niraparib and the Bcl-2 inhibitor venetoclax in triple negative breast cancer and AML cell lines, respectively. In summary, we have identified TNBC, ovarian and small cell lung cancer cell lines as highly sensitive to SY-1365 in vitro and have observed substantial tumor growth inhibition in PDX models of TNBC. SY-1365 induced a distinct transcriptional response compared with other transcriptional inhibitors, with apoptotic and DNA damage pathways being central. Finally, these mechanism of action studies support a rationale for investigating combinations of SY-1365 with inhibitors of PARP and Bcl-2. Citation Format: Shanhu Hu, Nan Ke, Yixuan Ren, Sofija Miljovska, Nisha Rajagopal, Michael McKeown, David Orlando, Kevin Sprott, Yoon J. Choi, Eric Olson, Christian C. Fritz. SY-1365, a potent and selective CDK7 inhibitor, exhibits promising anti-tumor activity in multiple preclinical models of aggressive solid tumors [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 1151. doi:10.1158/1538-7445.AM2017-1151

Phase 1 trial of CA-170, a novel oral small molecule dual inhibitor of immune checkpoints PD-1 and VISTA, in patients (pts) with advanced solid tumor or lymphomas.

TPS3099 Background: Programmed-death 1 (PD-1) and V-domain Ig suppressor of T-cell activation (VISTA) are independent immune checkpoints that negatively regulate T-cell function and are implicated in various malignancies. Preclinical studies have demonstrated that dual blockade of these pathways is synergistic. CA-170 is a first-in-class oral small molecule that directly targets both PD-1/PD-L1 and VISTA pathways and has shown anti-tumor activity in multiple preclinical models. Methods: The dose escalation phase has a target enrollment of 50 pts with advanced solid tumors or lymphomas onto escalating doses; the first four single-pt cohorts are accelerated titration but then switch to 3+3 design. The dose expansion phase has a target enrollment of 250 pts with select tumor types known to be responsive to anti-PD-1/L1 inhibitors and/or known to express PD-L1 or VISTA. Key eligibility criteria include: age ≥ 18 years, ECOG ≤1, adequate organ function, and ineligible for/did not respond to standard therapy including anti-PD-1/L1 inhibitors, where available. Primary objectives of this first-in-human study: safety, maximum tolerated dose, and recommended phase 2 dose. Secondary objectives: pharmacokinetics (PK) and anti-tumor activity. Exploratory endpoints: biomarkers and pharmacodynamic (PD) effects, which include changes in immune cell and peripheral cytokine populations in tumor (IHC/mRNA) and blood (flow cytometry/mRNA). Oral CA-170 is administered once daily in 21-day cycles. Response will be evaluated every other cycle per RECIST (v1.1) and Immune-related Response Criteria or by Cheson criteria (2007). Patients who discontinue treatment for reasons other than progressive disease will be followed for progression-free survival. Serial plasma, blood, and tumor samples will be collected for PK and PD evaluation. Clinical trial identifier: Clinical trial information: NCT02812875.

The Applications of the Novel Polymeric Fluoropyrimidine F10 in Cancer Treatment: Current Evidence

F10 is a novel polymeric fluoropyrimidine drug candidate with strong anticancer activity in multiple preclinical models. F10 has strong potential for impacting cancer treatment because it displays high cytotoxicity toward proliferating malignant cells with minimal systemic toxicities thus providing an improved therapeutic window relative to traditional fluoropyrimidine drugs, such as 5-fluorouracil. F10 has a unique mechanism that involves dual targeting of thymidylate synthase and Top1. In this review, the authors provide an overview of the studies that revealed the novel aspects of F10's cytotoxic mechanism and summarize results obtained in preclinical models of acute myeloid leukemia, acute lymphocytic leukemia, glioblastoma and prostate cancer that demonstrate the strong potential of F10 to improve treatment outcomes.

RXDX-107, A Dodecanol Alkyl Ester of Bendamustine, Demonstrates Greater Stability and Broad Antitumor Activity in Multiple Pre-Clinical Models of Solid Tumor

Purpose: RXDX-107 is a dodecanol alkyl ester of bendamustine encapsulated in human serum albumin (HSA) to form nanoparticles. The anti-tumor activity of bendamustine in solid tumor malignancies has been less impressive, partially due to short half-life. RXDX-107 was designed to extend the half-life and improve tissue biodistribution over bendamustine, which may result in superior efficacy and tolerability in patients with solid tumors. Experimental Design: The anti-tumor activity of RXDX-107 was measured in cellular anti-proliferation assay, cell-line derived xenograft (CDX) models and patient-derived xenograft (PDX) models. The mechanism of action and pharmacodynamics properties were measured by comet assay. The tumor accumulation was measured by a novel LC-MS/MS method. Results: In in vitro anti-proliferative studies, RXDX-107 displayed dose-dependent cytotoxicity against multiple solid tumor cell lines. While the IC50 of RXDX-107 were comparable to those of bendamustine, RXDX-107 displayed more complete cell killing. RXDX-107 exhibited enhanced pharmacodynamics properties, including stronger induction of pH2AX (a biomarker for DNA damage) and higher interstrand crosslinks (ICLs) formation. RXDX-107 significantly reduces tumor growth in human NSCLC xenograft models. RXDX-107 also showed single agent anti-tumor activities, including tumor regression in multiple PDX models of solid tumors including breast, lung, and ovarian cancer. Furthermore, the mode of action data exhibit slow and sustained release of bendamustine from RXDX-107, and high intratumoral accumulation of RXDX-107. Conclusions: Our preclinical data demonstrate potent and broad anti-tumor activity of RXDX-107 across a variety of solid tumor types, and support further clinical development of this novel drug candidate for the treatment of solid tumors.

SGN-CD123A, a Pyrrolobenzodiazepine Dimer Linked Anti-CD123 Antibody Drug Conjugate, Demonstrates Effective Anti-Leukemic Activity in Multiple Preclinical Models of AML

Survival expectations for acute myeloid leukemia (AML) patients remain poor, highlighting the need for further treatment options. The majority of AML blasts express CD123, the alpha subunit of the IL-3 receptor, which regulates the proliferation, survival and differentiation of hematopoietic cells. CD123 is also robustly expressed on leukemic stem cells and is a marker for minimal residual disease (MRD, Roug et al. 2012). Poor prognosis has previously been associated with elevated expression of CD123 on leukemic stem cells and blasts (Vergez et al. 2011, Testa et al. 2002). These findings identify CD123 as a rational therapeutic target in AML.Here we report the preclinical development of a novel CD123-directed immunoconjugate SGN-CD123A, consisting of a humanized anti-CD123 antibody conjugated to a highly potent DNA binding pyrrolobenzodiazepine (PBD) dimer drug via a protease-cleavable dipeptide linker. An engineered cysteine on each heavy chain attaching the PBD dimer to the antibody allows uniform drug loading of approximately two PBD dimers per antibody. Fluorescence microscopy studies show that SGN-CD123A is rapidly internalized and traffics to lysosomes within hours of binding to CD123-positive AML cells. Uptake of the antibody-drug-conjugate (ADC) induced DNA damage as measured by dose-and time-dependent increases in the phosphorylation of histone 2AX (γH2AX) and cell death associated with G2-M cell cycle arrest, caspase-3 activity, formation of cleaved poly ADP-ribose polymerase, and DNA fragmentation in target cells.The anti-leukemic activity of SGN-CD123A was assessed in cytotoxicity assays in 12 AML cell lines and 23 primary AML patient samples with variable cytogenetic abnormalities (favorable, intermediate and adverse) and multi-drug resistance (MDR) status. SGN-CD123A was highly active in 11 of 12 AML cell lines tested (mean IC50, 6 ng/ml; range of 0.02 to 38 ng/ml), including 4 of 5 MDR-positive cell lines, whereas it was inactive in CD123-negative HEL92.1.7 AML cells. SGN-CD123A was also active against 20 of 23 primary samples isolated from AML patients (mean IC50 of responsive samples, 0.8 ng/mL; range of 0.06 to 2.5 ng/ml). In both AML panels, molecular abnormalities, including the presence of a p53 mutation, FLT3-ITD, as well as MDR positivity, did not affect the in vitro cytotoxic activity of SGN-CD123A.In vivo antitumor activity was evaluated in AML xenograft models established with CD123-positive, MDR-negative Molm-13, HNT-34, and THP-1 cell lines and the MDR-positive KG-1 cell line. In all of the in vivo models, a single dose of SGN-CD123A delivered significant antitumor activity. SGN-CD123A dosed once at 10 mcg/kg yielded complete cures and significant survival advantage in the Molm-13 disseminated model of AML (p < 0.0001 compared to untreated or control ADC groups). Durable complete regressions were observed with a single dose of 25 or 75 mcg/kg in the MDR-negative HNT-34 subcutaneous model (p =0.0019 to control ADC group). In the THP-1 model, a single 100 mcg/kg dose of SGN-CD123A yielded durable complete regressions in 2 of 8 mice (p=0.0003 to untreated) whereas a higher dose of 300 mcg/kg gave complete tumor regressions in all mice (p < 0.0001 to untreated group). SGN-CD123A was also effective in a MDR-positive model of AML. A single dose of 100 mcg/kg SGN-CD123A significantly decreased tumor growth (p=0.003 to controls) whereas a single dose of 300 mcg/kg yielded durable complete regressions compared to the control groups in the KG-1 subcutaneous model of MDR-positive AML (p =0.008). Early evidence of the antitumor activity of SGN-CD123A was found in tumors harvested from THP-1 mice. Within 48h of dosing with SGN-CD123A, tumor cells showed elevated levels of the DNA damage marker γH2AX and changes in nuclear morphology.These data demonstrate that SGN-CD123A exhibits significant antitumor activity against a broad panel of primary AML samples and in preclinical models of MDR-positive AML that are characteristically resistant to chemotherapy. CD123-directed delivery of PBD may represent a promising new approach for the treatment of AML. DisclosuresSutherland:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc: Employment, Equity Ownership. Walter:Seattle Genetics, Inc: Consultancy, Research Funding. Westendorf:Seattle Genetics, Inc: Employment. Valliere-Douglass:Seattle Genetics, Inc: Employment. Pan:Seattle Genetics, Inc: Employment. Sussman:Seattle Genetics, Inc: Employment. Anderson:Seattle Genetics, Inc: Employment. Zeng:Seattle Genetics, Inc: Employment. Stone:Seattle Genetics, Inc: Employment. Klussman:Seattle Genetics, Inc: Employment. Ulrich:Seattle Genetics, Inc: Employment. Jonas:Seattle Genetics, Inc: Employment. Senter:Seattle Genetics, Inc: Employment. Drachman:Seattle Genetics, Inc: Employment. Benjamin:Seattle Genetics, Inc: Employment.

ALK2 Inhibition Via TP-0184 Abrogates Inflammation-Induced Hepcidin Expression and Is a Potential Therapeutic for Anemia of Chronic Disease

Hepcidin, a key liver peptide hormone, is essential to the regulation of bioavailable iron and erythropoiesis. Activin-like kinase receptor 2 (ALK2) signaling, via SMAD transcription factors, plays an important part in the regulation of hepcidin expression induced by pro-inflammatory cytokines. In chronic inflammatory conditions, such as rheumatoid arthritis, chronic kidney disease, colitis, and in some forms of cancer, hepcidin expression is induced. This induction of hepcidin expression results in lower levels of bioavailable iron, ultimately leading to the onset of anemia. Hepcidin regulates bioavailable iron levels by binding to and inhibiting the cellular iron pump, ferroportin. Ferroportin is important to macrophage-based iron recycling and dietary iron absorption. Several reports have suggested that lowering hepcidin provides a novel approach for targeting the clinical challenge of anemia. Currently approved approaches for these patients rely on transfusions and the use of erythropoietin-based therapies. Unfortunately, neither of these approaches address the underlying chronic inflammation or hepcidin induction and resulting anemia. In this report, we validate our small molecule inhibitor of ALK2, TP-0184, for the treatment of hepcidin-driven anemia of chronic diseases. Biochemical assays demonstrate that TP-0184 inhibits of the kinase activity of ALK2 with an IC50 of 5 nM. In vitro, TP-0184 is effective at targeting hepcidin expression with an EC50 lower than 100 nM in HepG2 cells. Three in vivo models were also explored for our validation of TP-0184. In our first study, turpentine oil TO was injected into the intrascapular fat pad of C57BL/6 mice to induce an acute inflammatory response that results in hepcidin-driven anemia. The animals were dosed with TP-0184 1 hour prior to TO treatment and once again 8 hours later. The TO-mediated acute inflammatory response in mice resulted in a 14-fold increase in liver hepcidin levels. Two oral doses of TP-0184 at 100 mg/kg, separated by 8 hours, reversed the induction of hepcidin that followed TO treatment. TP-0184 was tested at multiple doses in which efficacy was observed. In our second in vivo model, we induced anemia via intraperitoneal injection with heat-inactivated Brucella abortus. The mice were treated daily with TP-0184 for 3-7 days, after which, whole blood, plasma and livers were collected, from which liver and plasma hepcidin, plasma iron, and complete blood counts were assessed. Treatment with 100 mg/kg TP-0184 completely abrogated the Brucella abortus-induced reduction of hemoglobin and total red blood cell counts. In our third in vivo study, TP-0184 was also evaluated in the TC-1 lung cancer model for cancer-induced anemia. TC-1 tumor-bearing animals exhibited a 3-fold increase in liver hepcidin levels, which was reversed by dosing with 25 mg/kg TP-0184. From these experiments, we conclude that TP-0184 is a potent and selective inhibitor of ALK2 with demonstrated activity in multiple preclinical models of anemia associated with inflammation. TP-0184 also demonstrates favorable pharmacokinetic properties as well as good drug-like qualities, making it a strong candidate molecule with which to move into IND-enabling studies and formal clinical development. The current study supports a clinical development approach focused on anemia of chronic disease where an erythropoietin-sparing approach might offer significant clinical benefit to patients. DisclosuresPeterson:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

Abstract B173: Galeterone shows anti-tumor activity in multiple pre-clinical models that express androgen receptor splice variants, supporting correlative patient data seen in ARMOR2

Abstract Multiple experimental approaches were used to examine whether and by what mechanism galeterone affects prostate cancer growth in cells and tumors expressing androgen receptor (AR) splice variants, such as AR-V7. Galeterone is a selective, multitargeted, small molecule that disrupts androgen signaling at multiple points in the pathway. ARMOR3-SV is a Phase 3, randomized, open-label, multicenter, controlled clinical trial in metastatic castration resistant prostate cancer (mCRPC) patients whose tumors express AR splice variant-7 mRNA (AR-V7). AR-V7 is a truncated, constitutively active splice variant of the AR that lacks the ligand binding domain (LBD) and has been implicated in prostate cancer progression. The expression of AR-V7 occurs in approximately 14-26% of men with mCRPC prior to second-generation anti-androgens or chemotherapy, and AR-V7 expression has been clinically associated with resistance to enzalutamide (Xtandi) or abiraterone (Zytiga). Here we provide the pre-clinical and clinical rationale for the development of galeterone in mCRPC patients with AR-V7. Our findings show that using multiple pre-clinical approaches, galeterone reduces in vitro prostate cancer cell proliferation, androgen receptor signaling and xenograft tumor growth using cells and tumors that express AR splice variants. Reductions in AR splice variant protein were also observed in cells that also co-express wild-type AR, and prostate cancer cells that lack wild type AR, but transiently express AR splice variant protein. Clinical data further support activity of galeterone in patients with truncated androgen receptors. Galeterone's unique mechanism of AR protein downregulation occurs in both splice variant and wild-type AR, supporting that galeterone-induced AR downregulation is independent of the AR LBD. Because preclinical and clinical data support an opportunity for treatment in this setting, Tokai Pharmaceuticals designed ARMOR3-SV to test galeterone vs. enzalutamide in men with metastatic castration-resistant prostate cancer whose tumors express the AR-V7 splice variant. Citation Format: Douglas B. Jacoby, Amina Zoubeidi, Eva B. Corey, Elahe Mostaghel, Andrew K. Kwegyir-Afful, Senthilmurugan Ramalingam, Vincent Njar. Galeterone shows anti-tumor activity in multiple pre-clinical models that express androgen receptor splice variants, supporting correlative patient data seen in ARMOR2. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B173.

Phase 2 Trial of PRM-151, an Anti-Fibrotic Agent, in Patients with Myelofibrosis: Stage 1 Results

Phase 2 Trial of PRM-151, an Anti-Fibrotic Agent, in Patients with Myelofibrosis: Stage 1 Results

Editorial

<h3>Background</h3> PD-1/PD-L1 immune checkpoint inhibitors have transformed oncology, but a significant unmet need persists for patients with relapsed/refractory tumors following PD-1/PD-L1 treatment. PD-L1 is expressed in patients across a broad spectrum of tumor types and displays limited normal tissue expression, highlighting the potential of PD-L1 as a target for antibody-drug conjugates (ADCs) in addition to its role as an immune checkpoint. SGN-PDL1V is a PD-L1-directed ADC currently under preclinical investigation, which is comprised of an anti-PD-L1 antibody conjugated to the vedotin drug-linker. The vedotin drug-linker, consists of the microtubule disrupting agent, monomethyl auristatin E (MMAE), and a protease-cleavable peptide linker, which has been clinically validated in multiple ADC programs including brentuximab vedotin, enfortumab vedotin and polatuzumab vedotin.^1–3 The proposed SGN-PDL1V primary mechanism of action is direct cytotoxicity against PD-L1-expressing malignant cells through delivery of the MMAE payload. Additionally, MMAE induces immunogenic cell death, leading to subsequent immune activation in the tumor microenvironment.⁴ Here, we characterize the preclinical activity and tolerability of SGN-PDL1V. <h3>Methods</h3> SGN-PDL1V cytotoxicity was evaluated using PD-L1 expressing tumor cell lines in vitro and xenograft tumor models in vivo. Inhibition of the PD-1/PD-L1 immune checkpoint was assessed in a luminescent reporter system in vitro and a syngeneic tumor model in vivo. The tolerability and safety profile of SGN-PDL1V was determined in a non-human primate study. <h3>Results</h3> In vitro, SGN-PDL1V demonstrated internalization and potent cytotoxic activity against PD-L1 expressing tumor cells. In vivo, SGN-PDL1V achieved tumor regressions in multiple tumor xenograft models at doses as low as 1 mg/kg when dosed weekly for a total of three doses. This activity was observed in immunocompromised mice, which lack responses to PD-1/PD-L1 inhibition. Notably, activity was observed even in xenograft models with low, heterogeneous PD-L1 expression, supporting the possibility to treat patients across a wide range of PD-L1 expression levels. Additionally, SGN-PDL1V exhibited potential to inhibit the PD-1/PD-L1 checkpoint in vitro and in vivo. The tolerability and safety profile of SGN-PDL1V were assessed in a non-human primate study and found to be comparable to other FDA-approved vedotin ADCs. <h3>Conclusions</h3> SGN-PDL1V is a promising PD-L1 directed ADC with a unique cytotoxic mechanism of action among other PD-L1-targeted therapeutics. SGN-PDL1V demonstrated robust activity in multiple preclinical models and comparable tolerability and safety profile to other vedotin ADCs in non-human primates. Collectively, these data support further evaluation of SGN-PDL1V in a planned, first-in-human Phase 1 study. <h3>Acknowledgements</h3> We would like to thank Kerry Klussman for assay support and Jamie Mitchell for conjugation support. <h3>Trial Registration</h3> N/A <h3>References</h3> Senter PD, Sievers EL. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. <i>Nat Biotechnol</i> 2012;<b>30</b>(7):631–7. Epub 2012/07/12. doi: 10.1038/nbt.2289. PubMed PMID: 22781692. Rosenberg JE, O9Donnell PH, Balar AV, McGregor BA, Heath EI, Yu EY, et al. Pivotal trial of enfortumab vedotin in urothelial carcinoma after platinum and anti-programmed death 1/Programmed death ligand 1 therapy. <i>J Clin Oncol</i> 2019;<b>37</b>(29):2592–600. Epub 2019/07/30. doi: 10.1200/JCO.19.01140. PubMed PMID: 31356140; PubMed Central PMCID: PMC6784850. Tilly H, Morschhauser F, Bartlett NL, Mehta A, Salles G, Haioun C, et al. Polatuzumab vedotin in combination with immunochemotherapy in patients with previously untreated diffuse large B-cell lymphoma: an open-label, non-randomised, phase 1b-2 study. <i>Lancet Oncol</i> 2019;<b>20</b>(7):998–1010. Epub 2019/05/19. doi: 10.1016/S1470-2045(19)30091-9. PubMed PMID: 31101489. Klussman K, Tenn E, Higgins S, Mazahreh R, Snead K, Hamilton J, Grogan B, Sigurjonsson J, Cao A, Gardai S, Liu B. 618 Vedotin ADCs induce ER stress and elicit hallmarks of ICD across multiple cancer indications. <i>J Immunother Cancer</i> 2020;<b>8</b>(Suppl 3):A372. DOI:10.1136/jitc-2020-SITC2020.0618. <h3>Ethics Approval</h3> All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen or the external testing facility that conducted the studies.

E-3810 Is a Potent Dual Inhibitor of VEGFR and FGFR that Exerts Antitumor Activity in Multiple Preclinical Models

Tumor angiogenesis is a degenerate process regulated by a complex network of proangiogenic factors. Existing antiangiogenic drugs used in clinic are characterized by selectivity for specific factors. Antiangiogenic properties might be improved in drugs that target multiple factors and thereby address the inherent mechanistic degeneracy in angiogenesis. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family members and their cognate receptors are key players in promoting tumor angiogenesis. Here we report the pharmacologic profile of E-3810, a novel dual inhibitor of the VEGF and FGF receptors. E-3810 potently and selectively inhibited VEGF receptor (VEGFR)-1, -2, and -3 and FGF receptor (FGFR)-1 and -2 kinases in the nanomolar range. Ligand-dependent phosphorylation of VEGFR-2 and FGFR-1 was suppressed along with human vascular endothelial cell growth at nanomolar concentrations. In contrast, E-3810 lacked cytotoxic effects on cancer cell lines under millimolar concentrations. In a variety of tumor xenograft models, including early- or late-stage subcutaneous and orthotopic models, E-3810 exhibited striking antitumor properties at well-tolerated oral doses administered daily. We found that E-3810 remained active in tumors rendered nonresponsive to the general kinase inhibitor sunitinib resulting from a previous cycle of sunitinib treatment. In Matrigel plug assays performed in nude mice, E-3810 inhibited basic FGF-induced angiogenesis and reduced blood vessel density as assessed by histologic analysis. Dynamic contrast-enhanced magnetic resonance imaging analysis confirmed that E-3810 reduced the distribution of angiogenesis-sensitive contrast agents after only 5 days of treatment. Taken together, our findings identify E-3810 as a potent antiangiogenic small molecule with a favorable pharmacokinetic profile and broad spectrum antitumor activity, providing a strong rationale for its clinical evaluation.

Phase Ib dose-escalation study of MetMAb, a monovalent antagonist antibody to the receptor MET, in combination with bevacizumab in patients with locally advanced or metastatic solid tumors.

e13050 Background: Crosstalk between the Met and vascular endothelial growth factor (VEGF) pathways may be important during tumorigenesis. Aberrant activation of the HGF/Met pathway may promote angiogenesis via tumor cell secretion of angiogenic factors or directly activating endothelial cells. The combination of Met and VEGF inhibition resulted in enhanced antitumor activity in multiple preclinical models than either treatment alone. MetMAb is a recombinant, humanized, monovalent (one-armed) monoclonal antibody antagonist of HGF-induced Met signaling. In phase Ia, MetMAb was generally well tolerated up to 30 mg/kg IV Q3W. Herein, we describe results from a phase Ib study in which we tested MetMAb in combination with bevacizumab. Methods: The phase Ib trial tested the combination of MetMAb with bevacizumab in two cohorts: 3 patients in cohort 1 received MetMAb, 10 mg/kg, and bevacizumab, 15 mg/kg, IV Q3W; and 6 patients in cohort 2 received MetMAb,15 mg/kg, and bevacizumab,15 mg/kg IV Q3W. Pre- and post-dose sera were collected for evaluation of pharmacodynamic (PD) biomarkers that could be affected by inhibition of Met and/or VEGF signaling. Results: The combination of MetMAb with bevacizumab was generally well tolerated at all doses tested. No Gr3-5 drug- related toxicities were observed. One DLT of Gr1 hemoptysis in cohort 2 was observed in a patient who had central-necrosis of pulmonary metastases. Gr2 drug-related toxicities included peripheral edema and hypoalbuminemia. The most frequently observed toxicities (>30 %) included fatigue and increased weight. The best response was stable disease, with 3 patients receiving ≥ 6 cycles. PD biomarker data will be discussed. MetMAb PK was similar to that from phase Ia and no apparent interaction with bevacizumab was observed. Conclusions: This phase Ib trial demonstrated that the combination of MetMAb and bevacizumab is generally safe and well tolerated at the recommended dose of 15mg/kg Q3W for each agent. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Genentech