Bryan Oronsky

Dinitroazetidines Are a Novel Class of Anticancer Agents and Hypoxia-Activated Radiation Sensitizers Developed from Highly Energetic Materials

In an effort to develop cancer therapies that maximize cytotoxicity, while minimizing unwanted side effects, we studied a series of novel compounds based on the highly energetic heterocyclic scaffold, dinitroazetidine. In this study, we report the preclinical validation of 1-bromoacetyl-3,3-dinitroazetidine (ABDNAZ), a representative lead compound currently in a phase I clinical trial in patients with cancer. In tumor cell culture, ABDNAZ generated reactive free radicals in a concentration- and time-dependent manner, modulating intracellular redox status and triggering apoptosis. When administered to mice as a single agent, ABDNAZ exhibited greater cytotoxicity than cisplatin or tirapazamine under hypoxic conditions. However, compared with cisplatin, ABDNAZ was better tolerated at submaximal doses, yielding significant tumor growth inhibition in the absence of systemic toxicity. Similarly, when combined with radiation, ABDNAZ accentuated antitumor efficacy along with the therapeutic index. Toxicity studies indicated that ABDNAZ was not myelosuppressive and no dose-limiting toxicity was apparent following daily administration for 14 days. Taken together, our findings offer preclinical proof-of-concept for ABDNAZ as a promising new anticancer agent with a favorable toxicity profile, either as a chemotherapeutic agent or a radiosensitizer.

NO to cancer: The complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001.

The endogenous mediator of vasodilation, nitric oxide (NO), has been shown to be a potent radiosensitizer. However, the underlying mode of action for its role as a radiosensitizer – while not entirely understood – is believed to arise from increased tumor blood flow, effects on cellular respiration, on cell signaling, and on the production of reactive oxygen and nitrogen species (RONS), that can act as radiosensitizers in their own right. NO activity is surprisingly long-lived and more potent in comparison to oxygen. Reports of the effects of NO with radiation have often been contradictory leading to confusion about the true radiosensitizing nature of NO. Whether increasing or decreasing tumor blood flow, acting as radiosensitizer or radioprotector, the effects of NO have been controversial. Key to understanding the role of NO as a radiosensitizer is to recognize the importance of biological context. With a very short half-life and potent activity, the local effects of NO need to be carefully considered and understood when using NO as a radiosensitizer. The systemic effects of NO donors can cause extensive side effects, and also affect the local tumor microenvironment, both directly and indirectly. To minimize systemic effects and maximize effects on tumors, agents that deliver NO on demand selectively to tumors using hypoxia as a trigger may be of greater interest as radiosensitizers. Herein we discuss the multiple effects of NO and focus on the clinical molecule RRx-001, a hypoxia-activated NO donor currently being investigated as a radiosensitizer in the clinic.

Safety and activity of RRx-001 in patients with advanced cancer: a first-in-human, open-label, dose-escalation phase 1 study

BACKGROUND Epigenetic alterations have been strongly associated with tumour formation and resistance to chemotherapeutic drugs, and epigenetic modifications are an attractive target in cancer research. RRx-001 is activated by hypoxia and induces the generation of reactive oxygen and nitrogen species that can epigenetically modulate DNA methylation, histone deacetylation, and lysine demethylation. The aim of this phase 1 study was to assess the safety, tolerability, and pharmacokinetics of RRx-001. METHODS In this open-label, dose-escalation, phase 1 study, we recruited adult patients (aged >18 years) with histologically or cytologically confirmed diagnosis of advanced, malignant, incurable solid tumours from University of California at San Diego, CA, USA, and Sarah Cannon Research Institute, Nashville, TN, USA. Key eligibility criteria included evaluable disease, Eastern Cooperative Group performance status of 2 or less, an estimated life expectancy of at least 12 weeks, adequate laboratory parameters, discontinuation of all previous antineoplastic therapies at least 6 weeks before intervention, and no residual side-effects from previous therapies. Patients were assigned to receive intravenous infusions of RRx-001 at increasing doses (10 mg/m(2), 16·7 mg/m(2), 24·6 mg/m(2), 33 mg/m(2), 55 mg/m(2), and 83 mg/m(2)) either once or twice-weekly for at least 4 weeks, with at least three patients per dose cohort and allowing a 2-week observation period before dose escalation. Samples for safety and pharmacokinetics analysis, including standard chemistry and haematological panels, were taken on each treatment day. The primary objective was to assess safety, tolerability, and dose-limiting toxic effects of RRx-001, to determine single-dose pharmacokinetics, and to identify a recommended dose for phase 2 trials. All analyses were done per protocol. Accrual is complete and follow-up is still on-going. This trial is registered with ClinicalTrials.gov, number NCT01359982. FINDINGS Between Oct 10, 2011, and March 18, 2013, we enrolled 25 patients and treated six patients in the 10 mg/m(2) cohort, three patients in the 16·7 mg/m(2) cohort, three patients in the 24·6 mg/m(2) cohort, four patients in the 33 mg/m(2) cohort, three patients in the 55 mg/m(2), and six patients in the 83 mg/m(2) cohort. Pain at the injection site, mostly grade 1 and grade 2, was the most common adverse event related to treatment, experienced by 21 (84%) patients. Other common drug-related adverse events included arm swelling or oedema (eight [32%] patients), and vein hardening (seven [28%] patients). No dose-limiting toxicities were observed. Time constraints related to management of infusion pain from RRx-001 resulted in a maximally feasible dose of 83 mg/m(2). Of the 21 evaluable patients, one (5%) patient had a partial response, 14 (67%) patients had stable disease, and six (29%) patients had progressive disease; all responses were across a variety of tumour types. Four patients who had received RRx-001 were subsequently rechallenged with a treatment that they had become refractory to; all four responded to the rechallenge. INTERPRETATION RRx-001 is a well-tolerated novel compound without clinically significant toxic effects at the tested doses. Preliminary evidence of activity is promising and, on the basis of all findings, a dose of 16·7 mg/m(2) was recommended as the targeted dose for phase 2 trials. FUNDING EpicentRx (formerly RadioRx).

Preclinical Evaluation of the Metabolism and Disposition of RRx-001, a Novel Investigative Anticancer Agent

RRx-001 has shown promise as a novel cancer therapeutic agent. The disposition of RRx-001 was evaluated in vitro and after intravenous administration to rats. At both 24 and 168 h after a single intravenous administration of 14C-RRx-001 (10 mg/kg), the majority of radiolabel was in the blood. The recovery of label in excreta was quite low, but the major route of radiolabel excretion was via the kidney, with approximately 26% in the urine by the first 8 h and decreasing amounts in all subsequent collections to a total of 36.3% by 168 h. The partitioning of total radioactivity in red blood cells (RBCs) and plasma was determined after in vitro addition to human, rat, dog, and monkey whole blood at 1 and 20 μM. In rat, at 30 min, approximately 75% of the radioactivity is associated with RBCs and 25% with plasma. In human, at 30 min, approximately 25% of the radioactivity is associated with RBCs and 75% with plasma. Analysis by liquid chromatography/radiodetection/mass spectrometry showed that 14C-RRx-001 reacted rapidly with whole blood to give four major soluble metabolites: the GSH and Cys adducts of RRx-001 (M1 and M2) and the corresponding mononitro GSH and Cys adducts (M3 and M4). Human Hb was incubated with cold RRx-001 in buffer, and a standard proteomics protocol was used to separate and identify the tryptic peptides. Standard peptide collision-induced fragment ions supported the structure of the peptide GTFATLSELHCDK with the alkylation on the Cys-93 locus of the Hb β chain.

Two Case Reports of Resensitization to Previous Chemotherapy with the Novel Hypoxia-Activated Hypomethylating Anticancer Agent RRx-001 in Metastatic Colorectal Cancer Patients

The development of chemoresistance is a persistent problem during the treatment of cancer. Although reversion or modification of acquired chemoresistance has been previously observed, no systematic exploration has been undertaken. Here, we report a case study of 2 male patients, 62 and 66 years old, both with histologically proven, radiologically progressing, extensively pretreated, metastatic and refractory (≥2 conventional regimens and drug therapy) colorectal adenocarcinoma that was previously treated with FOLFIRI. The patients were resensitized to FOLFIRI after exposure to RRx-001 in the context of a phase-1 study. RRx-001 is a novel, hypomethylating and free-radical-inducing anticancer agent that activates nitrite reduction to NO under hypoxia and has an impact on epigenetic pathways. The repression of DNA methyltransferase 1 by RRx-001 may lead to demethylation and reexpression of silenced tumor suppressor genes, leading to resensitization. These examples provide insight into a nascent strategy to improve the prognosis in heavily pretreated cancer patients and suggest routes for further exploration.

Episensitization: Therapeutic Tumor Resensitization by Epigenetic Agents: A Review and Reassessment

Resistance to chemotherapy, biological and targeted therapies is an important clinical problem. Resistance can arise and/or be selected for multiple mechanisms of action. Unfortunately, acquired resistance to antitumor agents or regimens is nearly inevitable in all patients with metastatic disease. Until recently, it was believed that this resistance was unalterable and irreversible, rendering retreatment with the same or similar drugs futile in most cases. However, the introduction of epigenetic therapies, including HDAC inhibitors and DNA methyltransferase inhibitors (DNMTIs), has provided oncologists with new strategies to potentially overcome this resistance. For example, if chemoresistance is the product of multiple non-genetic alterations, which develop and accumulate over time in response to treatment, then the ability to epigenetically modify the tumor to reconfigure it back to its baseline non-resistant state, holds tremendous promise for the treatment of advanced, metastatic cancer. This minireview aims (1) to explore the potential mechanisms by which a group of small molecule agents including HDACs (entinostat and vorinostat), DNA hypomethylating agents such as the DNMTIs (decitabine (DEC), 5-azacytidine (5-AZA)) and redox modulators (RRx-001) may reprogram the tumors from a refractory to non-refractory state, (2) highlight some recent findings in this area, and (3) discuss the therapeutic potential of resensitization approaches with formerly failed chemotherapies.

Rewriting the Epigenetic Code for Tumor Resensitization: A Review

In cancer chemotherapy, one axiom, which has practically solidified into dogma, is that acquired resistance to antitumor agents or regimens, nearly inevitable in all patients with metastatic disease, remains unalterable and irreversible, rendering therapeutic rechallenge futile. However, the introduction of epigenetic therapies, including histone deacetylase inhibitors (HDACis) and DNA methyltransferase inhibitors (DNMTIs), provides oncologists, like computer programmers, with new techniques to “overwrite” the modifiable software pattern of gene expression in tumors and challenge the “one and done” treatment prescription. Taking the epigenetic code-as-software analogy a step further, if chemoresistance is the product of multiple nongenetic alterations, which develop and accumulate over time in response to treatment, then the possibility to hack or tweak the operating system and fall back on a “system restore” or “undo” feature, like the arrow icon in the Windows XP toolbar, reconfiguring the tumor to its baseline nonresistant state, holds tremendous promise for turning advanced, metastatic cancer from a fatal disease into a chronic, livable condition. This review aims 1) to explore the potential mechanisms by which a group of small molecule agents including HDACis (entinostat and vorinostat), DNMTIs (decitabine and 5-azacytidine), and redox modulators (RRx-001) may reprogram the tumor microenvironment from a refractory to a nonrefractory state, 2) highlight some recent findings, and 3) discuss whether the current “once burned forever spurned” paradigm in the treatment of metastatic disease should be revised to promote active resensitization attempts with formerly failed chemotherapies.

Epigenetic effects of RRx-001: a possible unifying mechanism of anticancer activity

RRx-001 is a novel aerospace-derived compound currently under investigation in several ongoing Phase II studies. In a Phase I trial, it demonstrated anti-cancer activity and evidence of resensitization to formerly effective therapies in heavily pre-treated patients with relapsed/refractory solid tumors. RRx-001 generates reactive oxygen and nitrogen species (ROS and RNS) and nitric oxide (NO), elicits changes in intracellular redox status, modulates tumor blood flow, hypoxia and vascular function and triggers apoptosis in cancer cells. We investigated the effect of RRx-001 on the epigenome of SCC VII cancer cells. RRx-001 at 0.5 and 2 μM significantly decreased global DNA methylation, i.e., 5-methylcytosine levels, in SCC VII cells. Consistently, 0.5-5 μM RRx-001 significantly decreased Dnmt1 and Dnmt3a protein expression in a dose- and time-dependent manner. In addition, global methylation profiling identified differentially methylated genes in SCC VII cells treated with 0.5, 2, and 5 μM RRx-001 compared to control cells. Twenty-three target sites were hypomethylated and 22 hypermethylated by >10% in the presence of at least two different concentrations of RRx-001. Moreover, RRx-001 at 2 μM significantly increased global acetylated histone H3 and H4 levels in SCC VII cells after 24 hour treatment, suggesting that RRx-001 regulates global acetylation in cancer cells. These results demonstrate that, in contrast to the traditional “one drug one target” paradigm, RRx-001 has multi(epi)target features, which contribute to its anti-cancer activity and may rationalize the resensitization to previously effective therapies observed in clinical trials and serve as a unifying mechanism for its anticancer activity.

A novel hypoxia-selective epigenetic agent RRx-001 triggers apoptosis and overcomes drug resistance in multiple myeloma cells

The hypoxic bone marrow (BM) microenvironment confers growth/survival and drug resistance in multiple myeloma (MM) cells. Novel therapies targeting the MM cell in its hypoxic BM milieu may overcome drug resistance. Recent studies led to the development of a novel molecule RRx-001 with hypoxia-selective epigenetic and nitric oxide-donating properties. Here, we demonstrate that RRx-001 decreases the viability of MM cell lines and primary patient cells, as well as overcomes drug resistance. RRx-001 inhibits MM cell growth in the presence of BM stromal cells. RRx-001-induced apoptosis is associated with: (i) activation of caspases; (ii) release of ROS and nitrogen species; (iii) induction of DNA damage via ATM/γ-H2AX; and (iv) decrease in DNA methyltransferase (DNMT) and global methylation. RNA interference study shows a predominant role of DNMT1 in MM cell survival versus DNMT3a or DNMT3b. The deubiquitylating enzyme USP7 stimulates DNMT1 activity, and conversely, USP7-siRNA reduced DNMT1 activity and decreased MM cell viability. RRx-001 plus USP7 inhibitor P5091 triggered synergistic anti-MM activity. MM xenograft studies show that RRx-001 is well tolerated, inhibits tumor growth and enhances survival. Combining RRx-001 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity. Our results provide the rationale for translation of RRx-001, either alone or in combination, to clinical evaluation in MM.

Episensitization: Defying Time’s Arrow

The development of cancer is driven by complex genetic and epigenetic changes that result in aberrant and uncontrolled cellular growth. Epigenetic changes, in particular, are implicated in the silencing or activation of key genes that control cellular growth and apoptosis and contribute to transformative potential. The purpose of this review is to define and assess the treatment strategy of “episensitization,” or the ability to sensitize cancer cells to subsequent therapy by resetting the epigenetic infrastructure of the tumor. One important facet is resensitization by epigenetic mechanisms, which goes against the norm, i.e., challenges the long-held doctrine in oncology that the reuse of previously tried and failed therapies is a clinically pointless endeavor. Thus, episensitization is a hybrid term, which covers recent clinically relevant observations and refers to the epigenomic mechanism of resensitization. Among the many formidable challenges in the treatment of cancer, the most inevitable is the development of acquired therapeutic resistance. Here, we present the basic principles behind episensitization and highlight the evidence suggesting that epigenetically mediated histone hypoacetylation and DNA hypermethylation events may reverse clinical drug resistance. The potential reversibility of epigenetic changes and the microenvironmental impact of epigenetic control on gene expression may mediate a return to a baseline state of treatment susceptibility. Episensitization is a novel and highly practical management strategy both to prevent the practice of permanent treatment discontinuation with the occurrence of resistance, which rapidly exhausts remaining options in the pharmaceutical armamentarium and to significantly extend patient survival. Accordingly, this review highlights several epigenetic agents including decitabine, vorinostat, entinostat, 5-azacitidine, oncolytic viruses, and RRx-001.