A novel retinoic acid receptor-γ agonist antagonizes immune checkpoint resistance in lung cancers by altering the tumor immune microenvironment.

All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA's anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA's anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice-a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.

A Novel CDK2/9 Inhibitor CYC065 Causes Anaphase Catastrophe and Represses Proliferation, Tumorigenesis, and Metastasis in Aneuploid Cancers.

Cyclin-dependent kinase 2 (CDK2) antagonism inhibits clustering of excessive centrosomes at mitosis, causing multipolar cell division and apoptotic death. This is called anaphase catastrophe. To establish induced anaphase catastrophe as a clinically tractable antineoplastic mechanism, induced anaphase catastrophe was explored in different aneuploid cancers after treatment with CYC065 (Cyclacel), a CDK2/9 inhibitor. Antineoplastic activity was studied in preclinical models. CYC065 treatment augmented anaphase catastrophe in diverse cancers including lymphoma, lung, colon, and pancreatic cancers, despite KRAS oncoprotein expression. Anaphase catastrophe was a broadly active antineoplastic mechanism. Reverse phase protein arrays (RPPAs) revealed that along with known CDK2/9 targets, focal adhesion kinase and Src phosphorylation that regulate metastasis were each repressed by CYC065 treatment. Intriguingly, CYC065 treatment decreased lung cancer metastases in in vivo murine models. CYC065 treatment also significantly reduced the rate of lung cancer growth in syngeneic murine and patient-derived xenograft (PDX) models independent of KRAS oncoprotein expression. Immunohistochemistry analysis of CYC065-treated lung cancer PDX models confirmed repression of proteins highlighted by RPPAs, implicating them as indicators of CYC065 antitumor response. Phospho-histone H3 staining detected anaphase catastrophe in CYC065-treated PDXs. Thus, induced anaphase catastrophe after CYC065 treatment can combat aneuploid cancers despite KRAS oncoprotein expression. These findings should guide future trials of this novel CDK2/9 inhibitor in the cancer clinic.

Perturbed Vitamin A Status Induced by Iron Deficiency Is Corrected by Iron Repletion in Rats with Pre-Existing Iron Deficiency.

BACKGROUND: Although iron deficiency is known to interrupt vitamin A (VA) metabolism, the ability of iron repletion to restore VA metabolism and kinetics in iron-deficient rats is not well understood. OBJECTIVES: In the present study, we examined the effects of dietary iron repletion on VA status in rats with pre-existing iron deficiency. METHODS: Weanling Sprague-Dawley rats were fed a VA-marginal diet (0.35 mg retinol/kg diet) containing either a normal concentration of iron [35 ppm, control group (CN)] or reduced iron (3 ppm, iron-deficient group, ID-); after 5 wk, 4 rats/group were killed for baseline measurements. A 3H-labeled retinol emulsion was administered intravenously to the remaining rats (n = 6, CN; n = 10, ID-) as tracer to initiate the kinetic study. On day 21 after dosing, n = 5 ID- rats were switched to the CN diet, generating an iron-repletion group (ID+). Blood samples were collected at 34 time points ≤92 d after dose administration, when all rats were killed and iron and VA status were determined. RESULTS: At baseline, ID- rats had developed iron deficiency, with a reduced plasma VA concentration (0.67 compared with 1.20 µmol/L in ID- and CN rats, respectively; P < 0.01) and a tendency toward higher liver VA (265 compared with 187 nmol in ID- and CN rats, respectively; P = 0.10). On day 92, iron deficiency persisted in ID- rats, accompanied by 2-times higher liver VA (456 nmol compared with 190 nmol in ID- and CN rats, respectively; P < 0.001) but lower plasma VA (0.64 compared with 0.94 µmol/L in ID- and CN rats, respectively; P = 0.05). ID+ rats not only recovered from iron deficiency, but also exhibited less liver VA sequestration (276 nmol) and normal plasma VA (0.91 µmol/L, not different from CN rats). CONCLUSIONS: Our results suggest that iron repletion can remove the inhibitory effect of iron deficiency on hepatic mobilization of VA and restore plasma retinol concentrations in iron-deficient rats, setting the stage for kinetic studies of VA turnover in this setting.

Dietary Iron Repletion Stimulates Hepatic Mobilization of Vitamin A in Previously Iron-Deficient Rats as Determined by Model-Based Compartmental Analysis.

BACKGROUND: Iron deficiency can result in hyporetinolemia and hepatic vitamin A (VA) sequestration. OBJECTIVES: We used model-based compartmental analysis to determine the impact of iron repletion on VA metabolism and kinetics in iron-deficient rats. METHODS: At weaning, Sprague-Dawley rats were assigned to either a VA-marginal diet (0.35 mg retinol equivalent/kg) with adequate iron (35 ppm, control group [CN]) or reduced iron (3 ppm, iron-deficient group [ID-]), with an equivalent average body weight for each group. After 5 wk, n = 4 rats from each group were euthanized for baseline measurements of VA and iron indices, and the remaining rats (n = 6 CN, n = 10 ID-) received an intravenous injection of 3H-labeled retinol in an emulsion as tracer to initiate the kinetic study. On day 21 after dosing, half of the ID- rats were switched to the CN diet to initiate iron repletion, referred to as the iron-repletion group (ID+). From the time of dosing, 34 serial blood samples were collected from each rat over a 92-d time course. Plasma tracer and tissue tracee data were fitted to 6- and 4-compartment models, respectively, to analyze the kinetic behavior of VA in all groups. RESULTS: Our mathematical model indicated that ID- rats exhibited a nearly 6-fold decrease in liver VA secretion and >4-fold reduction in whole-body VA utilization, compared with CN rats, whereas these perturbed kinetic behaviors were notably corrected in ID+ rats, close to those from the CN group. CONCLUSIONS: Iron repletion can remove the inhibitory effect that iron deficiency exerts on hepatic mobilization of VA and restore retinol kinetic parameters to values similar to that of never-deficient CN rats. Together with improvements in iron and VA indices, our results suggest that restoration of an iron-adequate diet is sufficient to improve VA kinetics after a previous state of iron deficiency.

CYP26A1 gene promoter is a useful tool for reporting RAR-mediated retinoid activity.

Of numerous genes regulated by retinoic acid (RA), CYP26A1 is the most inducible gene by RA. In this study, we have used a shortened construct form, E4, of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase or red fluorescent protein (RFP) as the reporter gene and have tested its responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of RA in cells cotransfected with retinoic acid receptors. It also responded quantitatively to retinol and other retinoids. An isolated clonal line of HEK293T cells permanently transfected with the promoter driving the expression of RFP responded to both RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was used to assess the retinoid activity of 3 novel synthetic retinoid analogues, as well as of the intact serum samples of rats. Among the synthetic retinoid analogues tested, EC23 is more potent than RA at lower concentrations and was more stable than RA. The retinoid activities could be measured in control rat serum samples and were increased in the serum of RA-treated rats. This system offers a biologically-based alternative to mass-based retinoid analysis.