Overview Cancer cachexia is a wasting syndrome with extensive loss of skeletal muscle mass with or without adipose tissue. It may be contrasted with simple starvation in which fat replaces glucose as the preferred fuel to spare lean body mass. It results from altered metabolism rather than just an energy deficit, and cannot be reversed by conventional nutritional support. The causes of cancer‐related cachexia are multifactorial including production of procachectic cytokines and metabolic derangements. European cancer community is credited in developing new definition and classification of cancer cachexia. Recent studies identified anamorelin, an oral ghrelin analogue, as a new agent for the treatment of cancer cachexia.

Rigosertib (ON 01910.Na; RGS) is a clinical stage anticancer agent that causes spindle abnormalities and mitotic arrest in neoplastic cells. The drug inhibits PI3K and PLK1 signaling pathways, down regulates cyclin D1 expression and induces apoptosis. Previously, we reported identification of RPL18A (L18A), a protein from the large ribosomal subunit, as a putative binding target of RGS [Proc. AACR 2014, #4595]. Knock-down of L18A with siRNA caused apoptosis in cancer cell lines. Role of L18A in the function of the ribosome is not known. Goal of this study was to conduct structure-function analysis of L18A and create deficiency mutant(s) for future experiments. Human L18A is predicted to adopt the following linear order of secondary structural elements - alpha helices (a) and beta strands (b) - when assembled into the ribosome - b1b2a1b3b4b5b6a2a3b7a4 (from www.rcsb.org/pdb/protein/Q02543). The predicted 3D structure of L18A suggests overall bilobal architecture with a flanking C-terminal tail. Packed in tandem, each lobe contains one alpha helix surrounded by a beta sheet formed by 4 (N-lobe) or 3 (C-lobe) beta strands. To map the potential binding site for RGS, we engineered multiple mutants of L18A including truncations at its N and C termini. Our approach involved successive removal of the structural elements and a few substitutions with alanine. Mutant proteins were expressed in E.coli as GST fusions, and derivative cell lysates were tested in pull-down assay with biotin-conjugated RGS (RGSbio) and avidin beads. Pulled-down protein-drug complexes were analyzed by Western blot with anti-GST antibody. At the C-terminus, abolishing a4 helix (C - a4) preserved RGSbio-binding activity comparable to wild type (WT) L18A. Further C-truncations resulted in partial (C - a3b7a4) or complete loss of activity (C - a2→a4; C - b6→a4 and C - b5→a4; latter represents N-lobe while missing entire C-lobe). Unlike N-lobe, independently expressed C-lobe had specific binding activity, albeit half that of WT. At the N-terminus, stepwise truncation of amino acids 6 through 12 in the first beta strand (b1) resulted in gradual reduction of specific binding activity. To verify that this was not due to structure misfolding we tested mutants with single Ala substitutions in b1 and obtained similar results. Such mutants will serve as RGS-non-binding controls in future studies. In the model, L18A packs in the ribosome in a way that its surface at the side of two alpha helices of both lobes is engaged in protein-RNA interactions. However, the outer surface of the two cross-braced beta sheets in both lobes is completely exposed to solvent and interestingly, there is a cavity formed at the junction of two lobes that appears to be deep enough to accommodate one molecule of RGS. Taken together, our data suggest that rigosertib approaches RPL18A at the exposed hinge region between the two lobes and may thus perturb ribosomal function in cancer cells. Citation Format: Irina A. Oussenko, Yogesh K. Gupta, Rodrigo Vasquez-Del Carpio, M.V. Ramana-Reddy, Aneel K. Aggarwal, E. Premkumar Reddy, James F. Holland, Takao Ohnuma. Structure-function analysis of RPL18A, a putative binding target of rigosertib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 694. doi:10.1158/1538-7445.AM2015-694

Abstract Rigosertib (ON 01910.Na) is clinical stage anti-mitotic cancer inhibitor that causes spindle abnormalities in neoplastic cells. Rigosertib inhibits PI3K and PLK1 signaling pathways, down regulates cyclin D1 expression and induces apoptosis. Previously we reported rigosertib led to hyperphosphorylation of RanGAP1•SUMO1, regulator of RanGTP/RanGDP cycle, and hypothesized phosphatase inhibition [Cancer Res 2011; 71; 4968-76]. Using biotin-conjugated rigosertib and avidin-conjugated agarose beads, we sought rigosertib molecular target from HeLa cell lysates by pull-down assay. Long-linker 1910-biotin showed an IC50 for cytotoxicity similar to unmodified rigosertib and efficient avidin binding. Asynchronously grown HeLa cell lysates were incubated with designated ligands and avidin beads. Bound lysate proteins were recovered from beads, resolved on SDS-PAGE and stained. Specific protein band of ∼20 kDa was detected only with long-linker 1910-biotin. Identification of proteins from the gel was performed by LC-ESI-MS/MS. Four ribosomal proteins possessed Mascot score above cut-off level with RPL18A, score of 367, and other proteins scored 153 or less. Specific dose-dependent binding of long-linker 1910-biotin to RPL18A was confirmed by Western blot analysis in replicate pull-down experiments using HeLa or MOLT-3 cell lysates. Recombinant RPL18A possessed specific reactivity to 1910-biotin in the pull-down assay. Knock-down of RPL18A with siRNA causes apoptosis in cancer cell lines [Sudo et al, Genomics 2010; 95; 210-6; and our data]. We propose that nucleocytoplasmic transport of RPL18A is impaired when hyperphosphorylated RanGAP1•SUMO1 fails to provide RanGTP/GDP gradient for nuclear pore complex. Importin-9 (IPO-9) is a major nucleocytoplasmic transporter for RPL18A. Among other cargos of IPO-9 are HSP27 (important anti-apoptotic player), scaffold subunit A of PP2A (major mitotic phosphatase), core histones and ribosomal proteins. Considering the essential role of nucleocytoplasmic transport in general and probable consequences of impeded transport of HSP27, PP2A and/or core histones, that actually match those observed with rigosertib, we hypothesize that binding with RPL18A in the presence of hyperphosphorylated RanGAP1•SUMO1 interferes with normal function of IPO-9, resulting in a variety of malfunctions and apoptosis observed in rigosertib treated cancer cells. Citation Format: Irina Oussenko, Saikrishna Divakar, M. V. Ramana Reddy, James F. Holland, E. Premkumar Reddy, Takao Ohnuma. RPL18A as putative target of rigosertib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4595. doi:10.1158/1538-7445.AM2014-4595

Rigosertib (ON 01910.Na), a synthetic novel benzyl styryl sulfone, was administered to 28 patients with advanced cancer in a Phase I trial in order to characterize its pharmacokinetic profile, determine the dose-limiting toxicities (DLT), define the recommended phase II dose (RPTD) and to document any antitumor activity. Patients with advanced malignant neoplasms refractory to standard therapy were given escalating doses of rigosertib (50, 100, 150, 250, 325, 400, 650, 850, 1,050, 1,375, 1,700 mg/m2/24h) as a 3-day continuous infusion (CI) every 2 weeks. An accelerated Fibonacci titration schedule with specified decreases for toxicities was used for escalation until grade ≥2 toxicity occurred. Intrapatient dose escalation was allowed if toxicity was grade ≤2 and the disease remained stable. Plasma pharmacokinetics (PK) and urinary PK assessments were studied in the 1st and 4th cycles. Twenty-nine patients (12 men and 17 women; age 36-87 y with a median of 63 y) were registered, but one died before study drug was given. Twenty-eight patients received a median of 3 cycles of therapy. Most common grade ≥2 toxicities attributable to rigosertib included fatigue, anorexia, vomiting and constipation. DLTs included muscular weakness, hyponatremia, neutropenia, delirium and confusional state. Risk factors for severe toxicities include preexisting neurological dysfunction or advanced gynecologic cancer after pelvic surgery. Rigosertib pharmacokinetics showed rapid plasma distribution phases and urinary excretion. Elevations in plasma Cmax and AUC due to decreases in plasma clearance were associated with acute grade ≥3 toxicities. Of 22 evaluable patients, 9 (41%) achieved a best overall response of stable disease; all other patients (n=13; 59%) progressed. The median progression-free survival time was 50 days (95% confidence interval [CI]: 37-80 days). Nine (41%) patients survived for over 1 y. In summary, prolonged IV infusions of rigosertib were generally well tolerated. Nine (41%) patients achieved stable disease and 9 (41%) patients survived for over 1 year. The RPTD appears to be 850 mg/m2/24hr CI x 3 days. (ClinicalTrials.gov identifier: NCT01538537).

Abstract Rigosertib (ON 01910.Na, MW 473.5 Da), a novel synthetic benzylstyrylsulfone, was given to 27 patients with solid tumors in a Phase I clinical trial. Drug doses were 50 to 1,700 mg/m2/24 h x 3 days, continuously infused for 72 h (one cycle), repeated every 2 weeks. The drug was generally well tolerated. In isolated cases severe toxicities, including neuromuscular changes, small bowel obstruction and hyponatremia, were encountered. Urine samples were collected from 14 patients. For 12 patients, urine was collected only in the first cycle with the following doses: 400 mg/m2 - 2 patients, 850 mg/m2 - 1 patient, 1,050 mg/m2 - 3 patients, 1,375 mg/m2 - 5 patients, 1,700 mg/m2 - 1 patient. For two patients, urine was collected both in the 1st and 4th cycle: 1 patient with 325 mg/m2 in both cycles, and another patient, with 850 mg/m2 in the first cycle, and 1,050 mg/m2 in the fourth cycle. Urine collection periods were 0-4 h, 4-8 h, 8-24 h, 24-48 h, 48-72 h (end of infusion), 72- 96 h and 96-120 h (post-infusion). Urine was collected in 4 L bottles (volume recorded) and alkalinized with sodium bicarbonate to pH 8.0-9.0; 50 mL fractions were frozen at -80 °C, until analysis. Liquid chromatography-electrospray-mass spectrometry was used with selected reaction monitoring (SRM) of m/z transition from 474 to 216 for rigosertib and from m/z 379 to 120 for the internal standard (ON 01370, an analog compound, MW 378.4 Da). Transition of m/z from 394 to 162 was monitored to determine a possible metabolite (ON 01500, the parent compound). Urinary excretion of rigosertib was rapid in all patients, and ceased soon after the end of infusion. There were no cumulative effects in repeated collections in the fourth cycle. In terms of total drug recovered, the urinary excretion averaged 3.0% (0%-15%) in the 72-96 h collection interval and 0.14% (0-0.56%) in the 96-120 h interval. These results suggest that most drug excreted by 72 h. The amount of drug excreted ranged 3% to 15% of the total administered. Among 16 sets of urine samples (from 14 patients, including 2 patients with 2 sets each), at the dose of 325-400 mg/m2 (n=4) the level of urinary excretion ranged 3-9%; drug excretion was 8-15% at 850-1,050 mg/m2 (n=6), and at 1,375-1,700 mg/m2 levels the excretion was 7-15 % (n=6). Clinical toxicities did not appear to have influenced urinary excretion patterns. No significant amount of ON 01500 (a possible metabolite) was found in any urine sample examined; the detection limit was ≤ 10 ng/mL. These results are consistent with and complementary to previous reports: strong (up to 98%) protein binding of rigosertib in human plasma (Cancer Chemother. Pharmacol. 65:177, 2009), and discontinuous albumin binding of the drug (Drug Metab. Dispos. 38:1480, 2010), suggesting that high protein binding of rigosertib likely have influenced active tubular absorption and resulted in low levels of urinary excretion. Citation Format: Sool Y. Cho, John Roboz, Takao Ohnuma. Urinary excretion of rigosertib (ON 01910.Na), a novel synthetic benzylstyrylsulfone, in a phase I trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1171. doi:10.1158/1538-7445.AM2013-1171

Rigosertib (ON 01910.Na), a synthetic novel benzyl styryl sulfone, was administered to 28 patients with advanced cancer in a Phase I trial in order to characterize its pharmacokinetic profile, determine the dose-limiting toxicities (DLT), define the recommended phase II dose (RPTD) and to document any antitumor activity. Patients with advanced malignant neoplasms refractory to standard therapy were given escalating doses of rigosertib (50, 100, 150, 250, 325, 400, 650, 850, 1,050, 1,375, 1,700 mg/m(2)/24h) as a 3-day continuous infusion (CI) every 2 weeks. An accelerated Fibonacci titration schedule with specified decreases for toxicities was used for escalation until grade ≥2 toxicity occurred. Intrapatient dose escalation was allowed if toxicity was grade ≤2 and the disease remained stable. Plasma pharmacokinetics (PK) and urinary PK assessments were studied in the 1st and 4th cycles. Twenty-nine patients (12 men and 17 women; age 36-87 y with a median of 63 y) were registered, but one died before study drug was given. Twenty-eight patients received a median of 3 cycles of therapy. Most common grade ≥2 toxicities attributable to rigosertib included fatigue, anorexia, vomiting and constipation. DLTs included muscular weakness, hyponatremia, neutropenia, delirium and confusional state. Risk factors for severe toxicities include pre-existing neurological dysfunction or advanced gynecologic cancer after pelvic surgery. Rigosertib pharmacokinetics showed rapid plasma distribution phases and urinary excretion. Elevations in plasma Cmax and AUC due to decreases in plasma clearance were associated with acute grade ≥3 toxicities. Of 22 evaluable patients, 9 (41%) achieved a best overall response of stable disease; all other patients (n=13; 59%) progressed. The median progression-free survival time was 50 days (95% confidence interval [CI]: 37-80 days). Nine (41%) patients survived for over 1 y. In summary, prolonged IV infusions of rigosertib were generally well tolerated. Nine (41%) patients achieved stable disease and 9 (41%) patients survived for over 1 year. The RPTD appears to be 850 mg/m(2)/24hr CI x 3 days. (ClinicalTrials.gov identifier: NCT01538537).

Abstract Previous work from our laboratory showed that tumor cells exposed to an anticancer mitotic inhibitor, regosertib (ON 01910.Na), resulted in G2/M arrest and hyperphosphorylation of RanGAP1·SUMO1 (HRGS). These findings suggest that regosertib may act either as an inhibitor of a RanGAP1·SUMO1 phosphatase or stimulant of a new kinase (Cancer Res 2011; 71; 4968-76). We further studied the relationship between dephosphorylation and deSUMOylation of HRGS. In the present study we used DU145 prostate cancer cell line. After exposure to one microM regosertib for 24 h, cells were collected and subjected, in equal portions, to lysis in different lysis buffers composed of basic lysis buffer and varied in supplemented inhibitors. Basic lysis buffer contained 10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton; 0.5% NP-40; 1 mM EGTA and 1 mM EDTA. Thus, prepared cell lysates were analyzed by Western blot for expression of phosphorylated and non-phosphorylated forms of RanGAP1, RanGAP1·SUMO1, PP1 and Cdc25C. Results: Roche protease inhibitor cocktail, PMSF (0.5 mM), NaF (1 mM) and Na3VO4 (1mM) as inhibitors added to the basic lysis buffer were unable to protect against loss of SUMO-group from RanGAP1·SUMO1 and, concomitantly, against de-phosphorylation of PP1 phosphatase at T-320. However, addition of N-ethylmaleimide (NEM, an inhibitor of deSUMOylation) as a single inhibitor supplement in the basic lysis buffer resulted in concentration-dependent (2.5 ∼ 40mM) inhibition of deSUMOylation of HRGS. Yet, even at lower NEM concentration RanGAP1, devoid of the SUMO-group, was still phosphorylated. This might be in correlation with our finding that even low concentration of NEM in the lysis buffer kept PP1 in phosphorylated state at T320 (which is inhibitory to PP1), preventing it from auto-dephosphorylation and re-activation. Also, neither regosertib (up to 100 microM) nor okadaic acid (OA, up to 1 microM), added to the basic lysis buffer as putative inhibitors, could prevent loss of SUMO group from HRGS and dephosphorylation of PP1 at T320. Regosertib in the lysis buffer did not prevent dephosphorylation of hyperphosphorylated Cdc25C (aka mitotic Cdc25C), while NEM and OA did (the latter implies that dephosphorylation was carried out by PP1 in the lysate). Conclusion: These data show that hyperphosphorylated RanGAP1·SUMO1 can be deSUMOylated, but remains phosphorylated. DeSUMOylation may be a prerequisite for RanGAP dephosphosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3797. doi:1538-7445.AM2012-3797

The benzyl styryl sulfone, ON 01910.Na, is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell lines. We examined the effect of ON 01910.Na on DNA damage– signaling molecules upstream of Cdc25C (Chk1, Chk2, and H2AX), as well as on Ran GTPase-activating protein 1 conjugated to small ubiquitin-related modifier 1 (RanGAP1 SUMO1), a mitosis coordinator. Prostate cancer, lymphoma, and leukemic cells were incubated with the drug for 4, 16, or 24 hours. Cell lysates were resolved on SDS-PAGE and analyzed by Western blot. Camptothecin and doxorubicin treatment caused activation/phosphorylation of DNA damage-responsive molecules by 4 hours, whereas ON 01910.Na did not do so. ON 01910.Na caused hyperphosphorylation of RanGAP1 SUMO1 within 4 hours that was sustained for more than 24 hours. Mild phosphorylation of Chk2 was observed only after 24-hour exposure, indicating that DNA damage response was not an initial effect of ON 01910.Na. MOLT-3 cells, synchronized by double-thymidine block, when released into a medium containing ON 01910.Na, accumulated mitotic cell number with a peak from 10 to 14 hours and remained near plateau for 20 hours, which corresponded with the time of RanGAP phosphorylation. ON 01910.Na had minimal effects on tubulin polymerization. These findings imply that ON 01910.Na neither induces DNA damage directly nor acts as a tubulin toxin. Its biological activity appears to rely on prolonged phosphorylation/hyperphosphorylation of RanGAP1 SUMO1. M-phase arrest and the consequent induction of apoptosis that follows could possibly be attributed to it. ON 01910.Na may act as an inhibitor of a RanGAP1 SUMO1 phosphatase or a stimulant of a new kinase. RanGAP1 SUMO1 appears to be a new target pathway for cancer chemotherapy. Cancer Res; 71(14); 1–9. 2011 AACR.

The benzyl styryl sulfone, ON 01910.Na, is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell lines. We examined the effect of ON 01910.Na on DNA damage-signaling molecules upstream of Cdc25C (Chk1, Chk2, and H2AX), as well as on Ran GTPase-activating protein 1 conjugated to small ubiquitin-related modifier 1 (RanGAP1·SUMO1), a mitosis coordinator. Prostate cancer, lymphoma, and leukemic cells were incubated with the drug for 4, 16, or 24 hours. Cell lysates were resolved on SDS-PAGE and analyzed by Western blot. Camptothecin and doxorubicin treatment caused activation/phosphorylation of DNA damage-responsive molecules by 4 hours, whereas ON 01910.Na did not do so. ON 01910.Na caused hyperphosphorylation of RanGAP1·SUMO1 within 4 hours that was sustained for more than 24 hours. Mild phosphorylation of Chk2 was observed only after 24-hour exposure, indicating that DNA damage response was not an initial effect of ON 01910.Na. MOLT-3 cells, synchronized by double-thymidine block, when released into a medium containing ON 01910.Na, accumulated mitotic cell number with a peak from 10 to 14 hours and remained near plateau for 20 hours, which corresponded with the time of RanGAP phosphorylation. ON 01910.Na had minimal effects on tubulin polymerization. These findings imply that ON 01910.Na neither induces DNA damage directly nor acts as a tubulin toxin. Its biological activity appears to rely on prolonged phosphorylation/hyperphosphorylation of RanGAP1·SUMO1. M-phase arrest and the consequent induction of apoptosis that follows could possibly be attributed to it. ON 01910.Na may act as an inhibitor of a RanGAP1·SUMO1 phosphatase or a stimulant of a new kinase. RanGAP1·SUMO1 appears to be a new target pathway for cancer chemotherapy.

e13584 Background: ON 01910.Na is a novel multitargeted inhibitor of several regulatory pathways including polo-like kinase 1 (Plk1) and PI-3 kinases, with synergistic nonclinical activity when combined with oxaliplatin, prompting phase I clinical evaluation. Methods: Patients with advanced cancer received escalating doses of ON 01910.Na as 24hr weekly continuous infusions. Oxaliplatin was administered biweekly as a 85mg/m2, 2 hr infusion. The ON 01910.Na dose cohorts ranged from 250-1,350 mg/m2 based on a modified Fibonacci escalation algorithm using the accelerated titration scheme (NCI). Intrapatient dose escalation was allowed. Results: Thirty patients (pts) (22 females; ages: 29-80 yrs received an overall mean 9.4 (range=1-30) number of weeks of combination therapy: ON 01910.Na mg/m2: 250 n=10; 650 n=9; 1,050 n=8; 1,350 n=3. No dose-limiting toxicity was observed in any patient. Grade 3+ toxicities included pain (3 pts), lethargy and weakness (1 pt), pneumonia (1 pt), hyponatremia (1pt), anemia (1pt), transient ischemic attack (1pt), vomiting (1pt), urinary tract infection (1pt), shortness of breath and myocardial infarction (1pt). Confirmed partial response was observed in 1 pt with chemotherapy- refractory ovarian cancer (duration = 11 wks) and in another pt with metastatic breast cancer (duration = 13 wks). Stable disease was observed in 4 pts with colon cancer (42 wks and 12 wks), ovarian and renal cancer (12 wks). Conclusions: ON 01910.Na administered as a 24hr infusion in combination with oxaliplatin was well tolerated and resulted in objective tumor responses in advanced ovarian, breast, colon and renal cancer.

INTRODUCTION: RanGAP1·SUMO1 is phosphorylated before nuclear envelope breakdown when cells enter mitosis. Previous work from our laboratory showed that a new anticancer antimitotic agent, ON 01910.Na (abbreviated 1910), a benzyl styryl sulfone analog, induced RGSH in all four human tumor cell lines tested. RGSH was not observed in the presence of 1911, inactive analog of 1910. However, RGSH was observed in the presence of tubulin depolymerizing agent nocodazole. Tubulin polymerization assay revealed that, unlike nocodazole, neither 1910 nor 1911 had effects on tubulin polymerization in vitro. In this study, we examined whether other tubulin agents induce RGSH and whether known mitotic phosphatases are involved. METHODS and RESULTS: We used two cell lines, MOLT-3 ALL and DU145 prostate cancer cell lines. We examined RGSH in the presence of known tubulin toxins including paclitaxel, vinblastine, vincristine, colchicine and nocodazole at a concentration of 1 µM in 4 h and 24 h. Doxorubicin and camptothecin served as negative controls. Following treatment with drugs, cells were analyzed by Western blotting. Drug concentrations that inhibited cell growth by 50% (IC50) or 90% (IC90) were determined in 3-day drug exposure experiments. All tubulin agents in the study were capable of inducing RGSH after 4h and 24 h exposure in both cell lines. The drug-induced RGSH was inhibited by caffeine, an ATM and ATR kinase inhibitor, and roscovitine, a Cdk kinase inhibitor. Okadaic acid (OA), known primarily as PP1 and PP2A phosphatase inhibitor, was also found to be capable of RGSH, therefore making it plausible by analogy that 1910 is RanGAP1·SUMO1 phosphatase inhibitor. The concentrations of 1910, OA or tubulin agents sufficient to elicit RGSH in 24 h, also caused 50 to 90% growth inhibition, respectively. 1910, as well as all tubulin agents in the study, induced phosphorylation of the phosphatase Cdc25C (aka “mitotic” Cdc25C) and down-regulation of interphase form of Cdc25C. CONCLUSION: 1910 and selected tubulin agents caused RGSH at IC50 – IC90 concentrations. Similar to tubulin agents, 1910 produced Cdc25C phosphorylation and down-regulation of its interphase form. OA inhibition of PP1 phosphatase and activation of RGSH suggest that 1910 could also serve as a phosphatase inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2639. doi:10.1158/1538-7445.AM2011-2639

Abstract INTRODUCTION: The benzyl styryl sulfone ON 01910. Na (abbreviated as 1910) is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell cultures. The compound is currently in Phase 1 and 2 trials. Available data show the drug produces three major abnormalities in tumor cell lines: (a) aberrant cell division, including irregular chromosomal segregation and cytokinesis; (b) G2/M arrest and apoptosis in many tumor cells; and (c) decreased expression of Cdc25C phosphatase. Early data suggested that 1910 was a PLK1 inhibitor. Subsequent studies did not confirm this, although 1910 was found to inhibit the PLK pathway. Precise mechanism of action continues to be investigated. METHODS and RESULTS: We assessed DNA damage checkpoints throughout the cell cycle and effects on signaling molecules upstream of Cdc25C following treatment of DU145 prostate cancer, Bel7404 hepatoma, U937 lymphoma or MOLT-3 ALL cells with 1910. Camptothecin (CPT) and doxorubicin (DOX) served as positive controls. Cell lysates after drug exposure for 4, 16 or 24 h were resolved on SDS-PAGE and analyzed by Western blot for Chk1, Chk2, ATM, RanGAP1•SUMO1 and their phosphorylated forms. CPT and DOX exposure resulted in activation/phosphorylation of DNA damage-responsive molecules Chk1, Chk2 and ATM by 4 h, whereas 1910 did not. However, hyperphosphorylation of RanGAP1•SUMO1 was observed within 4 h and sustained for more than 24 h during 1910 exposure. This was also seen with the anti-tubulin agent, nocodazole, but not with ON 01911, an inactive analog of 1910. Mild phosphorylation of Chk2 was observed only after 24 h exposure, suggesting that DNA damage response is not a primary effect of 1910. MOLT-3 cells, synchronized by double thymidine block, were released into medium supplemented with 1910, which resulted in peak accumulation of G2/M cells by 9-12 h. The G2/M cell fraction remained in plateau for more than 20 h. This timeframe correlated with the hyperphosphorylation of RanGAP1•SUMO1. Cleaved Lamin B, detected by 16 h and thereafter in 1910-containing release medium, confirmed an active apoptotic process during this period. MOLT-3 cells released into drug-free medium reached G2/M peak at 9-10 h and continued to cycle with synchronized transition into G1 at 15-16 h. RanGAP1•SUMO1 was not hyperphosphorylated and no cleaved Lamin B was detected. Tubulin polymerization assay revealed that 1910 and ON 01911 had little or no effect, whereas nocodazole inhibited the process. CONCLUSION: These findings show that 1910 is neither a direct DNA damage response inducer nor a tubulin toxin, and suggest that 1910 is an inhibitor of RanGAP1•SUMO1 phosphatase. Its mechanism of action appears to rely on prolonged hyperphosphorylation of RanGAP1•SUMO1, leading to G2/M arrest and induction of apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2500.

Abstract Introduction: ON 01910. Na is an anti-cancer agent with demonstrated activity against both solid tumors and hematological cancers. The purpose of this research was to evaluate the effect of dose and administration schedule on ON 01910. Na pharmacokinetics (PK) in advanced, heavily pre-treated solid tumor patients. Methods: Data was collected in three Phase I protcols conducted in the US and in India covering a wide range of doses and intraveous infusion schedules: Protocol 1 (50-1375 mg/m2/day over 72 h); Protocol 2 (250 – 4450 mg/m2/day over 24 h) and Protocol 3 (2400-3200 mg over 2, 4 or 8 h). In several patients, pharmacokinetics were evaluated for more than 1 dosing cycle. Plasma samples were collected pre-dose and up to 72 hours post-infusion. ON 01910. Na plasma levels were determined by a validated LC/MS/MS method. Results: Ninety-five data sets from 81 patients were evaluated in this study. ON 01910. Na showed biphasic elimination from the plasma, regardless of dose and administration schedule. The functional half-life of ON 01910. Na, estimated from the initial decline of plasma levels following infusion termination, was less than 2 hours. This was confirmed in data from patients receiving prolonged infusions as ON. 01910. Na approached steady state levels within several hours after dose initiation. As noted in the table below, ON. 01910. Na clearance was lower at higher drug dosing rates. There were no differences in drug pharmacokinetics among the infusion schedules. Conclusion: The pharmacokinetics of ON 01910. Na is dose dependent. A continuous IV infusion would be recommended to treat patients because of its short plasma half-life and rapid clearance. Systemic drug exposure is not affected by type of dosing (flat dosing vs. BSA adjusted). No significant differences were noted between the PK profiles of patients in the US centers and the patients in the India centers.Effect of Dosing Rate on Clearance of ON 01910. NaDosing Rate (mg/m2/hr)NClearance (L/hr/m2)0-25189.3 ± 4.725-501610.0 ± 5.050-75209.2 ± 3.175-10065.5 ± 2.6100-200126.2 ± 3.8200-40083.3 ± 1.6400-800112.5 ± 1.8800-125041.9 +/− 0.47 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2766.

Abstract Abstract 2944 Background: ON 01910.Na a novel benzyl styryl sulfone derivative is under clinical development in hematologic malignancies. It is a multi-kinase/PI3 kinase inhibitor that promotes G2/M arrest and selectively induces apoptosis in cancer cells. Leukemic cells exhibit significantly higher levels of sensitivity to ON 01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). Azacitidine (AzaC), is first line therapy for patients (pts) with higher-risk MDS and produces a response rate of 50%. Pts relapsed or refractory to hypomethylating based therapies have a poor prognosis and there are no accepted effective second line treatments, thus a need for new agents. Methods: A phase I/II study of ON 01910.Na is being conducted in pts with hematological malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks. A CBC is performed weekly and a bone marrow (BM) is performed at baseline and week 4, 8, and then q3 months thereafter. Pts with higher-risk disease had to have failed a hypomethylating agent. Results: Ten pts with MDS or AML relapsed/refractory to a hypomethylating agent have been treated with ON 01910.Na thus far (table 1). The study cohort comprised pts with a diagnosis (Dx) RAEB-2 (4 pts), RAEB-T (1 pt), and AML (5 pts) (median age of 75 years). Their cytogenetic profile included 1 pt with normal, 2 with intermediate (+8), and 7 pts with poor risk cytogenetics (monosomy 7 and/or complex). Patients were treated between 5 and 70 weeks. Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: Marrow CR (3), hematologic improvement (HI-P) (2); erythroid (1) platelet (1). An additional 2 pts had a >50% BM blast decrease from baseline but not to <5%. Thus, 5/10 (50%) demonstrate a bone marrow response. Survival of these pts was 7.3, 15.7, and 16.4 months; one patient remains on study 5+ months. Four of the five responders had MDS at the initiation of treatment: RAEB-2 (3), CMMoL (1), AML (1). Responders had monosomy 7 (2), trisomy 8 (1) and complex cytogenetics (2). One pt had an elimination of the MDS clone and the others had persistence of the abnormal karyotype throughout their treatment course. Five pts had SD without HI at 4 weeks, 2 pts progressed to AML. All 5 non-responders had AML; 4 with a proliferative course. These latter received only 2 (2) or 3 (3) cycles before succumbing to disease related infectious complications. Survival for these patients ranged from 1.3 – 2 months with a median duration on study of 42 days. The most frequent side effects grade2 2 for all pts included fatigue, anorexia, nausea, and dysuria in patients receiving extended duration infusions. One pt had a grade 3 urinary frequency. No hematologic toxicities occurred and no bone marrow toxicity or hypoplasia was noted. Pharmacokinetic studies are ongoing, data to date demonstrate no evidence of drug accumulation in patients who are treated repeatedly. Conclusion: ON 01910.Na appears to be safe and well tolerated in patients with refractory or relapsed MDS and AML. ON 01910.Na has biologic activity with reduction in BM blasts, eradication of the MDS clone and improvement in the peripheral blood counts in some pts. These effects are associated with increased survival albeit in limited numbers of pts treated thus far. Further study of ON 01910.Na is warranted to better define biologic activity, appropriate target populations and to define mechanism of action. Disclosures: Silverman: Onconova Therapeutics Inc: Research Funding, Research support of Clinical Trial. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership.

Sodium (E)-{N-[2-methyloxy-5-(2′,4′,6′-trimethoxy-styrylsulfonyl) methylenephenyl]amino}acetate (C21H24NNaO8S, ON 01910.Na) is a novel, synthetic benzyl styryl sulfone, currently in phase I clinical trials in cancer patients. Our objective was to use electrospray mass spectrometry to determine, in intact complexes, the number of drug molecules bound to albumin and selected enzymes. Native and recombinant albumin incubated with the drug, at various molar ratios, revealed simultaneous and discontinuous progression of drug binding, yielding intact albumin-drug complexes containing up to 22 drug molecules. Comparable complex protein-drug patterns were obtained for several enzymes, e.g., carbonic anhydrase. Intact albumin-ON 01910 complexes were also found in all patient samples. The drug-binding profiles were comparable, but not identical, for increasing sampling times and different doses (400–1700 mg/m2). We concluded that the techniques developed are capable of detecting the simultaneous formation of intact protein-drug complexes and of determining the number of drug molecules bound to proteins. The results enhance our hypothesis that drug binding may lead to conformational changes in proteins that, in turn, account for the exclusion of specific binding complexes and may influence protein behavior and activity. Application of these techniques reveals new insights about the nature of the antineoplastic drug ON 01910 in patient plasma, and the information obtained may have significance in understanding drug delivery to tumors.

Abstract Abstract 4839 Background ON 01910.Na, a novel benzyl styryl sulfone derivative is currently under clinical development as an anticancer agent in solid tumors and hematologic malignancies. ON 01910.Na selectively targets tumor cells at the mitotic phase of the cell cycle, arrests leukemia cells at the G2/M stage and induces apoptosis in a time and dose dependent manner. ON 01910.Na affects multiple cellular pathways, has multikinase activity and has been shown to target multiple genetic pathways, including those that are regulated by cyclin dependent kinases. Our studies indicate that leukemic cells exhibit significantly higher levels of sensitivity of ON01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). AzaC alters the natural history of higher risk MDS with effects on marrow function, reduction in the risk of leukemic transformation and increased overall survival (Silverman JCO 2002, 2006; Fenaux Lancet 2009). However, 50% of patients (pts) do not respond to AzaC and upon progression after treatment their prognosis is poor, thus, a need for alternative agents. Methods A phase I/II study of ON 01910.Na is being conducted in pts with heme malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CI) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks. Results This report details two pts with progressive disease following treatment for MDS with AzaC at a dose of 75mg/m2/d x 7 days SC every 4 weeks. Upon progression they were treated with ON 01910.Na at 1050 mg/m2/d CI x 72h to 144 hours. Patient 002 presented with MDS int-2 with myelofibrosis and mild splenomegaly, 8% marrow blasts, normal karyotype, and RBC transfusion dependence. AzaC therapy was initiated in 9/2004. A hematologic improvement (HI) was achieved according to modified IWG criteria with marrow myeloblasts < 5%, RBC transfusion independence and a persistence of mild splenomegaly. A stable HI was maintained for 4 years on monthly AzaC therapy until relapse. At progression (10/2008), marrow myeloblasts increased to 11%, RBC transfusion requirement resumed, platelets fell to 20 × 109/L, the spleen enlarged and a new cytogenetically abnormal clone emerged, 46,XY,del(20)(q11q13),i(21)(q10),der(21)t(1;21)(q12;p11) resulting in trisomy for 1q. Administration of ON 01910.Na 1050 mg/m2/d x 72 hours CI was initiated 3 months after the last dose of AzaC. After 2 cycles of treatment evaluation demonstrated a reduction in: marrow myeloblast to 1%, spleen size by 50% and the MDS clone by 74% by standard cytogenetic analysis. Following the 4th and 7th cycles of treatment with ON 01910.Na, marrow myeloblasts ranged from 1 to 3%, palpable splenomegaly resolved, RBC transfusions decreased by 33% and platelets rose to 80 × 109/L. Although the conventional karyotype normalized after the 4th cycle, all 3 abnormalites were detected by FISH in 30% of cells following the 4th and 7th cycles suggesting a loss of proliferative capacity of the MDS clone. Patient 004 presented with high risk MDS, 19% marrow blasts, pancytopenia and a complex karyotype. AzaC treatment was initiated in 9/2002 leading to a stable HI, with marrow myeloblasts < 5%, RBC transfusion independence, and persistence of the MDS clone with no change in the complex karyotype, including deletion of the MLL locus. The HI was maintained for 6.5 years with monthly AzaC therapy until progression. At relapse marrow myeloblasts increased to 19%, and the complex karyotype persisted unchanged from initial diagnosis. ON 01910.Na treatment started (3/2009) and evaluations at the 2nd, 4th and 7th cycles revealed: a) reduction in % marrow myeloblasts to 12%, 8% and 7% respectively; b) persistence of abnormal karyotype in over 90% of metaphase and interphase cells, irrespective of the decreased % blasts; c) increased platelets from 20 to 40 × 109/L. Patient 002 and 004 remain on treatment with ON 01910.Na at 8+ and 5+ months, respectively. Conclusions Preliminary data suggest that ON 01910.Na has an anti-proliferative modulatory effect on the MDS clone in pts progressing after AzaC therapy and should be explored in a broader MDS population. Disclosures Silverman: Onconova Therapeutics: Research Funding. Off Label Use: ON01910Na is investigational and not FDA approved. Wilhelm:Onconova: Employment. Reddy:Onconova Therapeutics Inc.: Consultancy, Equity Ownership, Grantee, Membership on an entity's Board of Directors or advisory committees. Holland:Onconova Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

In order to clarify cleavage efficiency of a multi-ribozyme expression system on their substrates,plasmids containing 20 cis-acting hammerhead ribozymes for self-cleavage and 10 trans-acting hammerhead ribozymes targeted on MDR1 and MRP1 substrates,the target plasmids pGEM-MDR1 and pGEM-MRP1 were constructed by employing PCR and DNA recombination.The endonuclease's digestion and DNA sequencing confirmed exactness of the multi-ribozyme expression systems and the target plasmids.The repeated self-cleavage tests revealed that the cisacting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and trans-acting hammerhead ribozymes 196 Rz and 210 Rz were released.The cleavage efficiencies of liberated ribozymes on their targets were evaluated in vitro and the results indicate that 196 Rz and 210 Rz were able to cleave the MDR1 and MRP1 RNA substrates.The profile of cleavage reaction shows that the cleavage efficiencies were correlated with the numbers of transacting hammerhead ribozymes contained in the multi-ribozyme expression systems,as well as the multi-ribozyme system is much better than the single ribozyme.Test of various concentrations of Mg2+ reveals that ribozyme cleavage reactions were dependent on Mg2+ concentration.The plot of lg(kobs) vs.lg[Mg2+] displays a linear relationship in the concentration range observed(2.5―20 mmol/L).

To better understand the cleavage efficiency of muhiribozyme system on its RNA substrate in the presence and absence of divalent magnesium and monovalent sodium ions.we constructed pGEM-Coat'A,pGEM-Coat'A196Rz plasmids and pGEM-MDRl target plasmid.They were applied to transcribe RNAs with SP6/T7 transcription kit.Cleavage reactions were carried out in cell-free system and reaction products were analyzed by electrophoresis on 6% denaturing polyacrylamide gels in TBS buffer.The gels were dried and exposed to X-ray films for autoradiography.The Image J software was employed to analyze the dried gels.The results indicated that the cleavage efficiency of the muhiribozyme was dependent on the concentration of divalent Mg2+.The cleavage products increased with the concentrations of divalent Mg2+ and were Mg2+ concentration and time dependent.No cleavage product was obtained in the presence of lower than 200 mmol/L Na+ alone.On the contrary,monovalent Na+ inhibited the Mg2+ -induced cleavage reaction in Na+ and Mg2+ coexistance.The cleavage rate was significantly lower than that observed with divalent Mg2+ alone.These results suggested that divalent Mg2+ was required for muhiribozyme on substrate cleavage reaction in the physical condition,whereas monovalent Na+ was not.