Anticancer Activity In Preclinical Models Research Articles

Abstract 2028: The triterpenoid CDDO-Methyl ester requires Nrf2 to decrease lung tumor burden and to protect against the toxicity of chemotherapy in experimental lung cancer

Abstract The NRF2 cytoprotective pathway, a drug target for many inflammation-related diseases, regulates immune cell function. While the anti-inflammatory nature of NRF2 activation protects healthy cells from malignant transformation, cancer cells can utilize the pathway to promote resistance to anti-cancer drugs. Up to 30% of human lung adenocarcinomas acquire mutations in either NFE2L2 or its negative regulator KEAP1 which result in constitutive activation of the NRF2 pathway. However, despite our knowledge of the important immunomodulatory effects of NRF2, these effects are not well characterized in the context of cancer. With NRF2 activators now approved for clinical use, it is critical to understand the impact of these drugs in cancer. Triterpenoids including CDDO-methyl ester (CDDO-Me, also known as bardoxolone methyl) are potent pharmacological NRF2 activators with demonstrated anti-cancer activity in preclinical models. In an early-stage preclinical model of lung cancer, CDDO-Me significantly decreased tumor burden in a dose- and Nrf2-dependent manner and improved immune signatures within the tumor microenvironment. However, most human lung cancers are not diagnosed until more advanced stages. To test CDDO-Me in an established tumor intervention model, lung tumors were initiated with vinyl carbamate in A/J WT and Nrf2 knockout (KO) mice. Tumors were allowed to develop for 8 weeks post initiation, after which mice were fed either a vehicle control diet or CDDO-Me (50-100 mg/kg of diet) for an additional 8-12 weeks, alone or in combination with carboplatin and paclitaxel (C/P) at 50 mg/kg and 15 mg/kg, respectively, by IP injection every other week. CDDO-Me significantly (p < 0.05) decreased surface lung tumor counts 35-71% in a Nrf2-dependent manner, and C/P significantly (p < 0.001) reduced tumor burden 53-59% regardless of Nrf2 status. The combination of CDDO-Me + C/P reduced tumor burden in WT lungs by 84% (p < 0.05), more than either agent alone. Nrf2 KO mice had an approximately two-fold increase (p < 0.001) of surface tumors compared to WT mice in both studies, regardless of treatment. C/P reduced tumor burden similarly in Nrf2 WT and Nrf2 KO mice. Interestingly, the combination of CDDO-Me + C/P increased T cell infiltration into the lungs of WT mice, but this change was not observed in groups treated with either agent alone. Importantly, CDDO-Me did not decrease the efficacy of C/P but protected WT mice from mortality and weight loss and lowered white and red blood cell counts. These studies suggest that NRF2 activation in advanced lung cancers can still decrease tumor burden by favorably modulating the immune microenvironment and, importantly, complements the anti-tumor efficacy of conventional chemotherapy while decreasing drug toxicity. Citation Format: Jessica Ann Moerland, Karen T. Liby. The triterpenoid CDDO-Methyl ester requires Nrf2 to decrease lung tumor burden and to protect against the toxicity of chemotherapy in experimental lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2028.

Inhibitors of NAD+ Production in Cancer Treatment: State of the Art and Perspectives.

The addiction of tumors to elevated nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of cancer metabolism. Obstructing NAD+ biosynthesis in tumors is a new and promising antineoplastic strategy. Inhibitors developed against nicotinamide phosphoribosyltransferase (NAMPT), the main enzyme in NAD+ production from nicotinamide, elicited robust anticancer activity in preclinical models but not in patients, implying that other NAD+-biosynthetic pathways are also active in tumors and provide sufficient NAD+ amounts despite NAMPT obstruction. Recent studies show that NAD+ biosynthesis through the so-called "Preiss-Handler (PH) pathway", which utilizes nicotinate as a precursor, actively operates in many tumors and accounts for tumor resistance to NAMPT inhibitors. The PH pathway consists of three sequential enzymatic steps that are catalyzed by nicotinate phosphoribosyltransferase (NAPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), and NAD+ synthetase (NADSYN1). Here, we focus on these enzymes as emerging targets in cancer drug discovery, summarizing their reported inhibitors and describing their current or potential exploitation as anticancer agents. Finally, we also focus on additional NAD+-producing enzymes acting in alternative NAD+-producing routes that could also be relevant in tumors and thus become viable targets for drug discovery.

MAP kinase ERK5 modulates cancer cell sensitivity to extrinsic apoptosis induced by death-receptor agonists

Death receptor ligand TRAIL is a promising cancer therapy due to its ability to selectively trigger extrinsic apoptosis in cancer cells. However, TRAIL–based therapies in humans have shown limitations, mainly due inherent or acquired resistance of tumor cells. To address this issue, current efforts are focussed on dissecting the intracellular signaling pathways involved in resistance to TRAIL, to identify strategies that sensitize cancer cells to TRAIL-induced cytotoxicity. In this work, we describe the oncogenic MEK5-ERK5 pathway as a critical regulator of cancer cell resistance to the apoptosis induced by death receptor ligands. Using 2D and 3D cell cultures and transcriptomic analyses, we show that ERK5 controls the proteostasis of TP53INP2, a protein necessary for full activation of caspase-8 in response to TNFα, FasL or TRAIL. Mechanistically, ERK5 phosphorylates and induces ubiquitylation and proteasomal degradation of TP53INP2, resulting in cancer cell resistance to TRAIL. Concordantly, ERK5 inhibition or genetic deletion, by stabilizing TP53INP2, sensitizes cancer cells to the apoptosis induced by recombinant TRAIL and TRAIL/FasL expressed by Natural Killer cells. The MEK5-ERK5 pathway regulates cancer cell proliferation and survival, and ERK5 inhibitors have shown anticancer activity in preclinical models of solid tumors. Using endometrial cancer patient-derived xenograft organoids, we propose ERK5 inhibition as an effective strategy to sensitize cancer cells to TRAIL-based therapies.

Abstract 5176: CDDO-methyl ester redirects macrophage polarization and reduces lung tumor burden in a Nrf2-dependent manner

Abstract The cytoprotective Nrf2 pathway impacts immune cell function and has been proposed as a target for many inflammation-related diseases, but the effects of Nrf2 activation on immune cells in the cancer context are not well characterized. With Nrf2 activators under development and in clinical trials, it is critical to understand the influence of these drugs on cancer progression. While the anti-inflammatory nature of Nrf2 activation protects healthy cells from malignant transformation, cancer cells can utilize the pathway to promote resistance to anti-cancer drugs and increase tumor cell survival. Up to 30% of human lung adenocarcinomas acquire mutations in the Nrf2 pathway which result in constitutive activation. However, triterpenoids, including CDDO-methyl ester (CDDO-Me, also known as bardoxolone methyl), are potent pharmacological Nrf2 activators with demonstrated anti-cancer activity in preclinical models. Lung cancer is the leading cause of cancer-related mortality worldwide, and macrophages are the most common immune cell type in the lung tumor microenvironment. To investigate Nrf2 activation in macrophages in the context of lung cancer, bone marrow-derived macrophages (BMDMs) were isolated from wild type (WT) and Nrf2 knock out (KO) mice and cultured in conditioned media from lung cancer cells to produce a tumor-educated phenotype. The triterpenoid CDDO-Me had anti-inflammatory effects in BMDMs stimulated with the conventional cytokines IFN-γ and LPS. Conversely, CDDO-Me increased (p < 0.05) the M1 macrophage markers TNFα, IL-6, and MHC-II and decreased the M2 macrophage markers VEGF, CCL2, and CD206 in tumor-educated BMDMs in a Nrf2-dependent manner. The phenotypic changes were observed on transcription, protein, and surface marker levels. This context-dependent reversal of BMDM polarization suggests that Nrf2 activation has different outcomes in different environments. To test CDDO-Me in vivo, lung tumors were initiated with vinyl carbamate in A/J WT and Nrf2 KO mice, and animals were fed either control diet or CDDO-Me (12.5-50 mg/kg of diet) for 16 weeks. CDDO-Me significantly (p < 0.05) decreased tumor number, size, and overall burden and reduced the histopathological severity of tumors in a Nrf2- and dose-dependent manner. Additionally, CDDO-Me increased the infiltration of CD64+ macrophages but decreased CD206+ expression in macrophages in the lungs of WT tumor-bearing mice. Interestingly, CD206 expression was higher on macrophages in the spleen of WT mice treated with CDDO-Me, suggesting a cancer context dependency. Future studies will evaluate the dependency of Nrf2 activation in macrophages for the anti-tumor activity of CDDO-Me. Citation Format: Jess Ann Moerland, Karen T. Liby. CDDO-methyl ester redirects macrophage polarization and reduces lung tumor burden in a Nrf2-dependent manner. [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 5176.

Antitumor activity of withaferin-A and propolis in benz (a) pyrene-induced breast cancer.

Female breast cancer is the leading malignancy surpassing lung cancer recently, and its incidence is continued to rise in many countries. The existing anticancer drugs have limitations like drug resistance and adverse effects leading to poor clinical outcomes. The natural compounds withaferin-A and propolis have been individually reported for their anticancer activity in preclinical models. However, the combined effect of these compounds has not been studied especially in breast cancer models. Therefore, it is of interest to evaluate the effect of Withaferin-A and propolis on Benz(a)pyrene-induced breast cancer. Wistar rats of female gender were treated with saline (normal control), Benz(a)pyrene (disease control), Benz(a)pyrene+ Withaferin-A or Propolis, Benz(a)pyrene+ Withaferin-A+ Propolis. At the end of the treatment, the plasma levels of carcino embryonic antigen (CEA) were measured. We observed a decrease in carcino embryonic antigen (CEA) levels in rats received withaferin-A and propolis combination rather than individual compounds indicating their beneficial role in breast cancer. Results of the present study show that propolis, when combined with withaferin A, exhibits better anti tumor activity than its individual effect in Benz (a) pyrene-induced mammary carcinogenesis.

Abstract P1-17-12: Preliminary data from a phase I/II, multicenter, dose escalation study of OP-1250, an oral CERAN/SERD, in subjects with advanced and/or metastatic estrogen receptor (ER)-positive, HER2-negative breast cancer

Abstract Background: ER+, HER2- metastatic breast cancer (MBC) patients will receive sequential, endocrine based therapy, either as monotherapy or in combination with a targeted agent until endocrine resistance develops. OP-1250 is a small molecule Complete Estrogen Receptor Antagonist (CERAN) that completely inactivates ER by blocking the 2 activation functions of ER transcription (AF-1 & AF-2). While complete antagonism is believed to be the most critical mechanism of action, OP-1250 is also a strong degrader of the ER. OP-1250 demonstrates anti-cancer activity in preclinical models, including activity against brain metastases and in tumors with activating mutations in ESR1. OP-1250 is orally bioavailable with favorable pharmacokinetics (PK) yielding high and steady drug levels in multiple species. The characteristics of OP-1250 including PK, ability to cross the blood brain barrier, and complete antagonism of the ER suggest that OP-1250 may have superior efficacy over standard of care and experimental agents, both as a monotherapy and in combination with other targeted therapies. Methods: OP-1250-001 is a phase I/II first-in-human study to determine the Dose Limiting Toxicity (DLT), Maximum Tolerated Dose (MTD) and/or Recommended Phase II Dose (RP2D) and to characterize the safety, PK profile and preliminary efficacy in subjects with ER+, HER2- MBC. Eligible patients received prior endocrine therapy. OP-1250 was given orally, once a day continuously in 28-day cycles. Using a rolling 6 design, cohorts of 3 - 6 subjects were sequentially enrolled and monitored for DLTs. The study allows for expansion of 1 or 2 doses for further evaluation of safety and PK to best inform selection of the RP2D. The phase II portion will evaluate the preliminary activity of OP-1250 in 3 cohorts: 1) measurable disease; and 2 exploratory cohorts: 2) evaluable & non-measurable; and 3) central nervous system metastasis. Plasma is being collected for ctDNA sequencing to evaluate ESR1 and other relevant mutations. Results: Between August 5, 2020 and June 4, 2021, 28 subjects with a median age of 63 (range 37-82) have been enrolled. Of the 27 subjects for whom detailed data on prior therapy was reported, all subjects received > 1 prior endocrine therapy for MBC (74% received > 2 and 37% received > 3; range 1-6). All of the 27 subjects received a CDK4/6 inhibitor and 23 of 27 (85%) had received fulvestrant. 20 subjects of 28 (71%) subjects had visceral disease affecting the liver, lung, peritoneum, and/or pleura. OP-1250 was escalated through 5 dose cohorts (range 30 - 300 mg qd). 26 patients were evaluable for a DLT. As of the data cut-off date, no DLTs occurred. Most of the TEAE were grade 1 or 2. OP-1250 is orally bioavailable and extensively distributed with an effective half-life of approximately 2-3 days. Cmax and AUC show dose proportional increase. By cycle 2, exposure levels are at steady state with limited fluctuations and at all doses significantly higher than those seen with fulvestrant. Importantly, OP-1250 has now demonstrated clinical activity at doses that were well tolerated and within the predicted exposure windows where maximum efficacy was observed in preclinical models. Detailed response and safety data will be presented. Conclusion: OP-1250 is continuing evaluation in a phase I/II study. The population enrolled reflects the evolving standard of care in that all pts received prior CDK4/6 inhibitors and the majority also received fulvestrant. OP-1250 is well tolerated with a favorable PK across dose levels ensuring target coverage throughout the dosing interval. No MTD has been identified and selection of the RP2D will be based on consideration of long-term tolerability, efficacy, and the ability to combine with targeted agents. (NCT04505826) Citation Format: Manish Patel, Carlos Alemany, Zahi Mitri, Della Makower, Virginia Borges, Joseph Sparano, Trinh Le, Pamela Klein, Julia Lawrence, Peter Kushner, Demiana Faltaos, Cyrus Harmon, David Myles, JoAnne Zujewski, Erika Hamilton. Preliminary data from a phase I/II, multicenter, dose escalation study of OP-1250, an oral CERAN/SERD, in subjects with advanced and/or metastatic estrogen receptor (ER)-positive, HER2-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-17-12.

AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.

AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, invitro and invivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

Abstract P237: PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification

Abstract Cyclin-dependent kinase 9 (CDK9) is a master regulator of transcription that controls paused RNA polymerase II (RNAP2) release through phosphorylation of its carboxy-terminal domain, resulting in productive transcription elongation. CDK9 has been extensively studied as a potential target for cancer therapy in “transcriptionally addicted” tumors as transient inhibition of CDK9 primarily depletes proteins with short half-lives, such as the oncogenes MCL1 and MYC, making CDK9 a promising target in cancer. Here we show that PRT2527 is a potent and highly selective CDK9 inhibitor with moderate to high clearance that achieves optimal temporal target engagement and exhibits potent in vitro and in vivo activities. PRT2527 inhibited CDK9 enzymatic activity with an IC50 of 0.98 nM in a biochemical assay and showed high selectivity in a panel of kinases when tested at physiologically relevant 1 mM ATP concentration. In vitro, PRT2527 inhibited phosphorylation of Ser2RNAP2 in NCI-H929 cells with an IC50 of 54 nM, and an IC50 of 198 nM in a plasma assay to adjust for human plasma protein binding. Transient treatment of cells with PRT2527 inhibited pSer2RNAP2, depleted MCL1 and MYC proteins, and activated cleaved caspase-3 (CC3) in a concentration-dependent manner. In a proteomic profiling study, MCL1 was identified as one of the major down-regulated proteins following PRT2527 treatment. In a panel of hematological cancer cell lines representing B- and T-ALL, AML, and non-Hodgkin’s lymphoma (NHL), as well as subsets of sarcoma, prostate, adenoid cystic carcinoma (ACC), and non-small cell lung cancer (NSCLC) cell lines, PRT2527 treatment consistently led to a potent, concentration-dependent inhibition of proliferation. In a pharmacokinetic/pharmacodynamic (PK/PD) study, intravenous (IV) administration of PRT2527 achieved transient target engagement, depletion of MCL1 and MYC proteins, and induction of apoptosis in tumor tissue. This PK/PD correlation was successfully translated into in vivo efficacy in multiple models. Once weekly dosing of PRT2527 was well-tolerated and significantly inhibited tumor growth in various AML CDX models and induced tumor regressions in double-hit and triple-hit diffuse large B-cell lymphoma (DLBCL) CDX and PDX models carrying the MYC translocation. Combining PRT2527 with venetoclax achieved complete tumor regressions in a venetoclax resistant OCI-AML3 model. PRT2527 demonstrated potent ex vivo activity in PDX models of B-ALL and T-ALL, as well as various solid tumor PDX models with high levels of MYC amplification and overexpression, including pancreatic carcinoma, gastric and gastroesophageal carcinomas, NSCLC, and sarcoma. In vivo efficacy studies with once weekly IV administration of PRT2527 confirmed significant tumor growth inhibition in select MYC-amplified solid tumor PDX models. Taken together, this preclinical characterization supports the advancement of PRT2527 into clinical studies for transcriptionally addicted hematological malignancies and solid tumors with MYC amplification and/or dysregulation. Citation Format: Yang W. Zhang, Liang Lu, Min Wang, Dave Rominger, Stefan Ruepp, Kirsten Gallagher, William Gowen-MacDonald, Chaofeng Dai, Miles Cowart, Andrew Combs, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. PRT2527 is a potent and selective CDK9 inhibitor that demonstrates anti-cancer activity in preclinical models of hematological malignancies and solid tumors with MYC amplification [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P237.

Reversal of Bortezomib-Induced Neurotoxicity by Suvecaltamide, a Selective T-Type Ca-Channel Modulator, in Preclinical Models.

Simple SummaryChemotherapy-induced peripheral neurotoxicity (CIPN) is a side-effect of anti-cancer medications, which can lead to pain, disruptions to movement, and eventually results in the need to interrupt or stop chemotherapy. This study sought to test whether the drug suvecaltamide could help to reduce the impact of the chemotherapy agent bortezomib (BTZ) on symptoms of CIPN using animal models and human cells. Suvecaltamide did reverse negative changes in nerve conduction velocity and intraepidermal nerve fiber density indicative of CIPN in rats, and did not interfere with the anti-cancer effect of BTZ. These results indicate that suvecaltamide could potentially be useful for patients experiencing CIPN, although further mechanistic and molecular studies in vitro and in vivo are required before clinical trials.This study evaluated suvecaltamide, a selective T-type calcium channel modulator, on chemotherapy-induced peripheral neurotoxicity (CIPN) and anti-cancer activity associated with bortezomib (BTZ). Rats received BTZ (0.2 mg/kg thrice weekly) for 4 weeks, then BTZ alone (n = 8) or BTZ+suvecaltamide (3, 10, or 30 mg/kg once daily; each n = 12) for 4 weeks. Nerve conduction velocity (NCV), mechanical threshold, β-tubulin polymerization, and intraepidermal nerve fiber (IENF) density were assessed. Proteasome inhibition was evaluated in peripheral blood mononuclear cells. Cytotoxicity was assessed in human multiple myeloma cell lines (MCLs) exposed to BTZ alone (IC50 concentration), BTZ+suvecaltamide (10, 30, 100, 300, or 1000 nM), suvecaltamide alone, or vehicle. Tumor volume was estimated in athymic nude mice bearing MCL xenografts receiving vehicle, BTZ alone (1 mg/kg twice weekly), or BTZ+suvecaltamide (30 mg/kg once daily) for 28 days, or no treatment (each n = 8). After 4 weeks, suvecaltamide 10 or 30 mg/kg reversed BTZ-induced reduction in NCV, and suvecaltamide 30 mg/kg reversed BTZ-induced reduction in IENF density. Proteasome inhibition and cytotoxicity were similar between BTZ alone and BTZ+suvecaltamide. BTZ alone and BTZ+suvecaltamide reduced tumor volume versus the control (day 18), and BTZ+suvecaltamide reduced tumor volume versus BTZ alone (day 28). Suvecaltamide reversed CIPN without affecting BTZ anti-cancer activity in preclinical models.

Pyrvinium Pamoate Induces Death of Triple-Negative Breast Cancer Stem-Like Cells and Reduces Metastases through Effects on Lipid Anabolism.

Cancer stem-like cells (CSC) induce aggressive tumor phenotypes such as metastasis formation, which is associated with poor prognosis in triple-negative breast cancer (TNBC). Repurposing of FDA-approved drugs that can eradicate the CSC subcompartment in primary tumors may prevent metastatic disease, thus representing an effective strategy to improve the prognosis of TNBC. Here, we investigated spheroid-forming cells in a metastatic TNBC model. This strategy enabled us to specifically study a population of long-lived tumor cells enriched in CSCs, which show stem-like characteristics and induce metastases. To repurpose FDA-approved drugs potentially toxic for CSCs, we focused on pyrvinium pamoate (PP), an anthelmintic drug with documented anticancer activity in preclinical models. PP induced cytotoxic effects in CSCs and prevented metastasis formation. Mechanistically, the cell killing effects of PP were a result of inhibition of lipid anabolism and, more specifically, the impairment of anabolic flux from glucose to cholesterol and fatty acids. CSCs were strongly dependent upon activation of lipid biosynthetic pathways; activation of these pathways exhibited an unfavorable prognostic value in a cohort of breast cancer patients, where it predicted high probability of metastatic dissemination and tumor relapse. Overall, this work describes a new approach to target aggressive CSCs that may substantially improve clinical outcomes for patients with TNBC, who currently lack effective targeted therapeutic options. SIGNIFICANCE: These findings provide preclinical evidence that a drug repurposing approach to prevent metastatic disease in TNBC exploits lipid anabolism as a metabolic vulnerability against CSCs in primary tumors.

Synergistic anticancer activity of sorafenib, paclitaxel, and radiation therapy on anaplastic thyroid cancer in vitro and in vivo.

This study aimed to investigate the antitumor activity of paclitaxel with radiation and sorafenib in anaplastic thyroid cancer (ATC) cells in vitro and in vivo. The 8505C ATC cell line was exposed to radiation, sorafenib, and paclitaxel each or in combination. The effects of combined treatment on the cell cycle and intracellular signaling pathways were assessed using flow cytometry and western blot analysis. An ATC cell line xenograft model was used to examine antitumor activity in vivo. Radiation, paclitaxel plus sorafenib synergistically decreased cell viability in ATC cells and significantly increased apoptotic cell death. The combination of paclitaxel, sorafenib with radiation reduced the antiapoptotic factor in ATC. This combination therapy significantly reduced the tumor volume and increased survival in the ATC xenograft model. These results suggest that the combination of radiation and paclitaxel plus sorafenib has significant anticancer activity in preclinical models.

Drugging "undruggable" genes for cancer treatment: Are we making progress?

RAS, TP53 (p53) and MYC are among the most frequently altered driver genes in cancer. Thus, RAS is the most frequently mutated oncogene, MYC the most frequently amplified gene and TP53 the most frequently mutated tumor suppressor gene and overall the most frequently mutated gene in cancer. Theoretically, therefore, these genes are highly attractive targets for cancer treatment. However, as the protein products of each of these genes lack an accessible hydrophobic pocket into which low molecular weight compounds might bind with high affinity, they have proved difficult to target and have traditionally been referred to as "undruggable." Despite this branding, several low molecular weight compounds targeting each of these proteins have recently been reported to have anticancer activity in preclinical models. Indeed, several drugs inhibiting mutant KRAS, MYC overexpression or reactivating mutant p53 have undergone or are currently undergoing clinical trials. For targeting mutant KRAS and reactivating mutant p53, trials have progressed to a Phase III stage, that is, the mutant-p53 reactivating drug, APR-246 is currently being investigated in patients with myelodysplastic syndrome (MDS) and the RAS inhibitor, rigosertib is also undergoing evaluation in patients with MDS. Although there appears to be no directly acting MYC inhibitor currently being tested in a clinical trial, an anti-MYC compound, known as OmoMYC has been extensively validated in multiple preclinical models and is being developed for clinical evaluation. Based on current evidence, the traditional perception of RAS, p53 and MYC as being "undruggable" would appear to be coming to an end.

Antibody Targeting of Eph Receptors in Cancer.

The Eph subfamily of receptor tyrosine kinases mediate cell-cell communication controlling cell and tissue patterning during development. While generally less active in adult tissues, they often re-emerge in cancers, particularly on undifferentiated or progenitor cells in tumors and the tumor microenvironment, associated with tumor initiation, angiogenesis and metastasis. Eph receptors are thus attractive therapeutic targets, and monoclonal antibodies have been commonly developed and tested for anti-cancer activity in preclinical models, and in some cases in the clinic. This review summarizes 20 years of research on various antibody-based approaches to target Eph receptors in tumors and the tumor microenvironment, including their mode of action, tumor specificity, and efficacy in pre-clinical and clinical testing.

A Combinatorial Strategy for Targeting BRAF V600E-Mutant Cancers with BRAFV600E Inhibitor (PLX4720) and Tyrosine Kinase Inhibitor (Ponatinib).

Most aggressive thyroid cancers are commonly associated with a BRAF V600E mutation. Preclinical and clinical data in BRAF V600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAF V600E thyroid cancer than in single-agent or BRAF and MEK inhibitors. The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs. The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAF V600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAF V600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control. Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAF V600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

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.

Targeted enhancement of the therapeutic window of L19-TNF by transient and selective inhibition of RIPK1-signaling cascade.

IntroductionCytokine-based products are gaining importance for cancer immunotherapy. L19-TNF is a clinical-stage antibody-cytokine fusion protein that selectively accumulates to tumors and displays potent anticancer activity in preclinical models. Here, we describe an innovative approach to transiently inhibit off-target toxicity of L19-TNF, while maintaining antitumor activity.MethodsGSK’963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice for its ability to reduce acute toxicity associated with TNF signaling. The biological effects of L19-TNF on tumor cells, lymphocytes and tumor vessels were investigated with the aim to enable the administration of TNF doses, which would otherwise be lethal.ResultsTransient inhibition of RIPK1 allowed to increase the maximal tolerated dose of L19-TNF. The protective effect of GSK’963 did not affect the selective localization of the immunocytokine to tumors as evidenced by quantitative biodistribution analysis and allowed to reach high local TNF concentrations around tumor blood vessels, causing diffused vascular shutdown and hemorrhagic necrosis within the neoplastic mass.ConclusionsThe selective inhibition of RIPK1 with small molecule inhibitors can be used as a pharmaceutical tool to transiently mask TNF activity and improve the therapeutic window of TNF-based biopharmaceuticals. Similar approaches may be applicable to other pro-inflammatory cytokines.

Abstract 4730: A novel RAD51 inhibitor, CYT01B, shows anti-cancer activity in preclinical models of AID expressing solid tumors

Abstract AID, a DNA-directed cytidine deaminase, plays a critical role in somatic hypermutation and immunoglobulin class switching in maturing B-lymphocytes. Unlike site specific recombinases such as RAG1/2, AID is a promiscuous DNA damaging enzyme that deaminates cytidines at sites throughout the genome. AID expressing cells become critically dependent on the homologous recombination factor RAD51 to repair DNA double strand breaks that result from these off target deamination events. We have developed a novel small molecule, CYT01B, which inhibits RAD51 response to DSBs and is potent to a much greater extent in AID expressing cells. Previously, we have shown this molecule to have activity in mouse lymphoma xenograft models that constitutively express AID. Here we present new preclinical characterization data of CYT01B in solid cancer models. We first analyzed the frequency and levels of AID overexpression in solid tumor data in The Cancer Genome Atlas. Multiple solid tumor types displayed ectopic AID expression, including breast cancer, sarcoma, melanoma, pancreatic cancer, lung cancer, and head and neck cancers with about 30% of the patient samples 4-fold above the baseline expression level. We then tested CYT01B in several solid tumor derived human cell lines. We observed a correlation between AID expression and activity. Cell lines with low ectopic expression of AID had EC50 values in the low micromolar range (~2µM), while those without AID expression gave EC50 values of about 5µM and higher. Next, we examined the activity of our small molecule in 14 different PDX models with various levels of AID expression. These models included renal, head and neck, lung, pancreatic, ovarian, colorectal, and breast cancer samples. We observed a wide range of tumor growth inhibition across the different models (0% to 60%+), which tended to correspond with AID expression; the higher the AID expression the greater the observed tumor growth inhibition. Taken together, these data demonstrate that CYT01B shows anti-cancer activity in a range of solid tumor models. Overall, this provides the basis for continued development of CYT01B as an AID/RAD51 synthetic lethal therapeutic for solid cancers. Citation Format: Melinda Day, Tyler Maclay, Kevin Mills. A novel RAD51 inhibitor, CYT01B, shows anti-cancer activity in preclinical models of AID expressing solid tumors [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 4730.

A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.

The Deubiquitinase Inhibitor b-AP15 and Its Effect on Phenotype and Function of Monocyte-Derived Dendritic Cells

The ubiquitin-proteasome system is elementary for cellular protein degradation and gained rising attention as a new target for cancer therapy due to promising clinical trials with bortezomib, the first-in class proteasome inhibitor meanwhile approved for multiple myeloma and mantle cell lymphoma. Both bortezomib and next-generation proteasome inhibitors mediate their effects by targeting the 20S core particle of the 26S proteasome. The novel small molecule inhibitor b-AP15 affects upstream elements of the ubiquitin-proteasome cascade by suppressing the deubiquitinase activity of both proteasomal regulatory 19S subunits and showed promising anticancer activity in preclinical models. Nonetheless, effects of inhibitors on the ubiquitin-proteasome system are not exclusively restricted to malignant cells: alteration of natural killer cell-mediated immune responses had already been described for drugs targeting either 19S or 20S proteasomal subunits. Moreover, it has been shown that bortezomib impairs dendritic cell (DC) phenotype and function at different levels. In the present study, we comparatively analyzed effects of bortezomib and b-AP15 on monocyte-derived DCs. In line with previous results, bortezomib exposure impaired maturation, antigen uptake, migration, cytokine secretion and immunostimulation, whereas treatment with b-AP15 had no compromising effects on these DC features. Our findings warrant the further investigation of b-AP15 as an alternative to clinically approved proteasome inhibitors in the therapy of malignancies, especially in the context of combinatorial treatment with DC-based immunotherapies.

Correction: Combined inhibition of PI3K and Src kinases demonstrates synergistic therapeutic efficacy in clear-cell renal carcinoma.

[This corrects the article DOI: 10.18632/oncotarget.25700.].