Inhibition of allogeneic inflammatory responses by the Ribonucleotide Reductase Inhibitors, Didox and Trimidox

<p>Abstract</p> <p>Background</p> <p>Graft-versus-host disease is the single most important obstacle facing successful allogeneic stem cell transplantation (SCT). Even with current immunosuppressive therapies, morbidity and mortality rates are high. Current therapies including cyclosporine A (CyA) and related compounds target IL-2 signaling. However, although these compounds offer great benefit, they are also associated with multiple toxicities. Therefore, new compounds with a greater efficacy and reduced toxicity are needed to enable us to overcome this hurdle.</p> <p>Methods</p> <p>The allogeneic mixed lymphocyte reaction (MLR) is a unique <it>ex vivo </it>method to study a drug's action on the initial events resulting in T-cell activation and proliferation, synonymous to the initial stages of tissue and organ destruction by T-cell responses in organ rejection and Graft-versus-host disease. Using this approach, we examined the effectiveness of two ribonucleotide reductase inhibitors (RRI), Didox and Trimidox, to inhibit T-cell activation and proliferation.</p> <p>Results</p> <p>The compounds caused a marked reduction in the proliferative responses of T-cells, which is also accompanied by decreased secretion of cytokines IL-6, IFN-γ, TNF-α, IL-2, IL-13, IL-10 and IL-4.</p> <p>Conclusions</p> <p>In conclusion, these data provide critical information to justify further investigation into the potential use of these compounds post allogeneic bone marrow transplantation to alleviate graft-versus-host disease thereby achieving better outcomes.</p

Targeting the Cell Cycle, RRM2 and NF-κB for the Treatment of Breast Cancers

A hallmark of cancer is the dysregulation of the cell cycle. The CDK4/6 inhibitor palbociclib is approved for treating advanced estrogen-receptor-positive breast cancer, but its success is limited by the development of acquired resistance owing to long-term therapy despite promising clinical outcomes. This situation necessitates the development of potential combination strategies. Here, we report that didox, an inhibitor of ribonucleotide reductase in combination with palbociclib, can overcome palbociclib resistance in ER-positive and ER-negative breast cancers. This study shows didox downregulates an element of the cell cycle checkpoint, cyclin D1, accompanied by a reduction in NF-κB activity in vitro and tumor growth inhibition of palbociclib-resistant ER positive breast cancer tumor growth in vivo. Furthermore, didox induces cell cycle arrest at G1 as well as reduces ROS generated by on-target effects of palbociclib on the cell cycle. Our current study also reports that the CCND1 and RRM2 upregulation associated with palbociclib-resistant breast cancers decreases upon ribonucleotide reductase inhibition. Our data present a novel and promising biomarker-driven combination therapeutic approach for the treatment of ER-positive and ER-negative breast cancers that involves the inhibition of the CDK4/6-cyclinD1/pRb cell cycle axis that merits further clinical investigation in human models

The efficacy of the ribonucleotide reductase inhibitor Didox in preclinical models of AML.

Acute Myeloid Leukemia (AML) is an aggressive malignancy which leads to marrow failure, and ultimately death. There is a desperate need for new therapeutics for these patients. Ribonucleotide reductase (RR) is the rate limiting enzyme in DNA synthesis. Didox (3,4-Dihydroxybenzohydroxamic acid) is a novel RR inhibitor noted to be more potent than hydroxyurea. In this report we detail the activity and toxicity of Didox in preclinical models of AML. RR was present in all AML cell lines and primary patient samples tested. Didox was active against all human and murine AML lines tested with IC50 values in the low micromolar range (mean IC50 37 µM [range 25.89-52.70 µM]). It was active against primary patient samples at concentrations that did not affect normal hematopoietic stem cells (HSCs). Didox exposure resulted in DNA damage and p53 induction culminating in apoptosis. In syngeneic, therapy-resistant AML models, single agent Didox treatment resulted in a significant reduction in leukemia burden and a survival benefit. Didox was well tolerated, as marrow from treated animals was morphologically indistinguishable from controls. Didox exposure at levels that impaired leukemia growth did not inhibit normal HSC engraftment. In summary, Didox was well tolerated and effective against preclinical models of AML

Didox has activity against AML in colony formation assays.

<p>A. Didox reduced colony formation in KG1a cells. Cells were exposed to titration of Didox for 24 hours before incubation in methylcellulose (7–8 days). Mean colony formation was assessed in triplicate in 3 experiments and normalized to untreated controls. B. Didox exposure reduced colony formation in primary AML samples. Primary samples (C1–C3) were exposed to a titration of Didox for 24 hours before incubation in methylcellulose (12–14 days). Mean colony formation was assessed in triplicate in 3 experiments and normalized to untreated controls. *  = p value less than 0.05.</p

Primary patient sample characteristics.

<p>Primary patient sample characteristics.</p

Didox has activity in AML models <i>in vivo</i>.

<p>A. Schema. 1.0×10⁶ luciferase tagged AML cells were injected into sublethally irradiated (4.5 Gy) recipients and allowed to engraft. Engraftment was monitored by bioluminescent imaging (IVIS 100 imager). Animals received 5 days of Didox at 425 mg/kg or D5 water control via intraperitoneal injection (IP). Animals were followed for survival. B. Representative bioluminescent images from Nras^G12D (MR2) mice pre- and post-treatment. C. Quantitation of bioluminescence post-treatment. D. Kaplan-Meier survival curves of Didox <i>in vivo</i> studies from start of treatment. *  = p of value less than 0.05.</p

MN1 overexpression and p53 knockdown induce resistance in AML <i>in vitro</i>.

<p>A. Confirmation of KD in M1p5 cells. B. M1p5 shP53 or GFP cells were exposed to a titration of Didox (0–25 µM) for 72 hours and viability assays performed. C. MFL2 shP53 or GFP cells were exposed to a titration of Didox (0–40 µM) for 72 hours and viability assays performed. D. 3 independent 72 hour viability assays with MN1 and GFP controls, in triplicate with titrations of Didox (0–50 µM).*  = p of value less than 0.05.</p

Inhibitory concentrations of murine and human AML.

<p>Inhibitory concentrations of murine and human AML.</p

Antimalarial Activities of Polyhydroxyphenyl and Hydroxamic Acid Derivatives

Several known mammalian ribonucleotide reductase inhibitors featuring a polyhydroxyphenyl and/or hydroxamate moiety as the active group were screened for potency in inhibiting growth of the malaria parasite Plasmodium falciparum. Compounds containing a 2,3- or 3,4-dihydroxyphenyl group as well as benzohydroxamate appear to be the most effective inhibitors of the malaria parasite

RR is expressed in AML.

<p>A. Western blots performed for RR small subunit. AML patient samples (bone marrow, M1–M3, leukopheresis A1–A5), and cell lines (K – KG1a, M – MFL2, H – HL-60, K5 – K562). B. Growth curves. Cell lines were treated with Didox for 72 hours. Viability was assessed and normalized to untreated controls.</p