Plinabulin was obtained from BeyondSpring Pharmaceuticals, Inc. (New York, NY, USA), irinotecan was obtained from Teva Pharmaceuticals (Irvine, CA, USA), docetaxel and paclitaxel were from Selleckchem (Houston, TX, USA). For in vivo studies, all compounds were diluted in 5% dextrose for intraperitoneal (i.p.) or intravenous (i.v.) injection of indicated doses. For in vitro studies, all drugs were diluted in complete growth media with 1% dimethyl-sulfoxide (DMSO). In vitro EGF treatment was performed at a concentration of 100 ng/ml.

Microtubule protein (MTP) preparations consisting of 70% tubulin and 30% microtubule-associated proteins were isolated from a bovine brain, polymerized into microtubules and monitored by light scattering at 350 nm. Transmission electron microscopy was used to determine the mean length distribution of microtubules in the absence or presence of the drug. In brief, samples were fixed at room temperature with 4% formaldehyde and 2.5% glutaraldehyde in 0.1 M PIPES buffer for 30 min and stained at room temperature for 7 min utilizing primary antibodies and for 2 min. with gold-labeled secondary antibodies. Embeding was performed at 60˚C with epoxy resin. Section thickness was 60 nm. Grids were viewed in a Jeol electron microscope-1200 EX11 (JEOL, Ltd., Tokyo, Japan) at x2,000 and x30,000 magnification. The Zeiss MOPIII was used to determine microtubule length distributions and mean lengths for at least 100 microtubules per sample.

All cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). HCT-15, A549 and MDA-MB-231 cells were maintained in RPMI-1640 medium (Lonza Group, Ltd., Basel, Switzerland). LoVo cells were maintained in F12-K medium (Corning/Cellgro; Corning Inc., Corning, NY, USA). DF1 cells were maintained in Gibco Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cell culture media were supplemented with 10% fetal bovine serum (FBS; Avantor, Inc., Radnor, PA, USA) and housed in a 5% CO₂ atmosphere at 37˚C, except the DF1 cells which were cultured at 39˚C.

A549 cells (1,000 cells/cm²) were seeded overnight on coverslips. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature, followed by permeabilization using 0.1% Triton in PBS for 5 min and blocking of unspecific binding with 5% donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) in PBS. These steps were done at room temperature and washing with PBS was done between any steps. Primary antibodies were rabbit anti-early endosomal antigen (EEA)1 (1:300; cat. no. 610456; BD Bioscences, San Jose, CA, USA) and mouse anti-KRAS (1:100; cat. no. ab172949; Abcam, Cambridge, MA, USA) and were applied overnight at 4˚C. After additional washing steps, cells were incubated with secondary donkey anti-rabbit-488 (1:200; cat. no. 711-545-152l; Jackson ImmunoResearch Laboratories, Inc.) and anti-mouse-Cy3 (1:200; cat. no. 715-165-150; Jackson ImmunoResearch Laboratories, Inc.) antibodies in PBS with 5% donkey serum for 30 min at room temperature, followed by washing and addition of Hoechst 33342 Solution (1 µg/ml; Thermo Fisher Scientific, Inc.) for 3 min, followed by washing, H₂O for 3 min, 100% ethanol for 3 min and air-drying prior to embedding (all steps at room temperature). Images were captured with a Zeiss LSM 780 NLO confocal microscope and quantification was performed using ImageJ, version 1.51 (National Institute of Health, Bethesda, MD, USA).

Indicated cell lines were grown in standard serum-containing media (RPMI with 10% FBS from American Type Culture Collection). Cell viability was measured in triplicate utilizing the adenosine triphosphate-based CellTiter-Glo^® assay (Promega Corporation, Madison, WI, USA).

Animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted by the U.S. National Institutes of Health. Animal research was prospectively approved by the Institutional Animal Care and Use Committee (Animal assurance institutional #A3226-01; Protocol #50842, Fred Hutchinson Cancer Research Center, Seattle, WA, USA). Female athymic Foxn1nu (BALB/c nude) mice between 4 to 6 weeks of age were obtained from Harlan Laboratories, Inc. (Madison, WI, USA). A total of 84 mice weighing 18-24 g and kept under germ-free conditions with a 12-h light/dark cycle at 20-24˚C with 40-60% humidity. For the HCT-15 and LoVo models, mice were anesthetized by isoflurane and inoculated with 0.1 ml of a 50% media/50% Matrigel mixture containing a suspension of cultured tumor cells (1x10⁷ cells/mouse for LoVo, 5x10⁶ cells/mouse for HCT-15). As the tumors grew, the length (largest diameter) and width (smallest diameter) of tumors were measured using a digital caliper to determine the mean radius for tumor volume estimates assuming a spherical geometry and calculation per V_tumor = 4/3 x π x r³. Automated calculation and documentation of tumor volume estimates was done using the animal study management software Study Director V.2.1.1 (Studylog Systems, Inc., San Francisco, CA, USA). When tumor volume estimates exceeded 50-60 mm³, mice were randomized into control and treatment groups, being pair-matched by tumor size, and dosing was initiated (Day 1). Mice were euthanized as described below.

A total of 40 male (n=23) or female (n=17) N/t-va;Ink4a/Arf^-/- agouti mice (Jackson Laboratory, Bar Harbor, ME, USA) were kept under germ-free conditions in a 12 h light/12 h dark cycle at 20-24˚C with 40-60% humidity. Mice were aged 6-8 weeks and weighed 18-24 g. These mice expressed the avian t-va receptor under control of the nestin promoter were injected intracranially with DF-1 cells (American Type Culture Collection) as hosts for the replicative avian retroviral gene transfer vector RCAS (Addgene, Inc., Cambridge, MA, USA). Tumors were generated utilizing two RCAS vectors for the expression of platelet-derived growth factor B (PDGFB) and either KRAS encoding the activating mutation KRAS^G12A, or green fluorescent protein (GFP). DF-1 cells infected with these RCAS strains were injected into the brains of adult mice (aged 4 to 6 weeks) under the anesthetic isoflurane. A total of 1 µl of a 1:1 mixture for a total combined injection of 4x10⁴ transfected DF-1 cell suspension was delivered using a 30-gauge needle attached to a Hamilton syringe. Coordinates were bregma 0 mm, lateral -0.5 mm (right of midline) and depth -1.5 mm from the dural surface. Mice were put on the study when they lost >0.3 g total over 2 consecutive days or displayed outward signs of a tumor. Mice (n=89) were treated with vehicle or plinabulin at 7.5 mg/kg twice per week for up to 10 weeks. The mice were euthanized in a carbon dioxide chamber (flow rate of CO₂ displacing 25% of the chamber volume per minute for 5 min total in accordance with American Veterinary Medical Association Euthanasia Guidelines of 2013) when they lost >20% of their body weight, displayed a lack of mobility, inability to feed or weighed <14 g for a male/12 g for a female. Animal death was confirmed following at least 1 min of immobility within the chamber, which was followed immediately by decapitation and harvesting of the brain tissue.

All statistical comparisons were performed using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA). Kaplan-Meier survival curves were prepared using and analyzed using the log-rank (Mantel-Cox) test. Time series data were analyzed using linear regression with the mean ± standard error calculated for each time-point. Direct comparisons were performed using a two-tailed t-test. P<0.05 was considered to indicate a statistically significant difference. All experiments were performed in triplicate unless stated otherwise.

The overlapping binding sites of plinabulin and colchicine at the α/β-tubulin interface led to characterizing and comparing the effects of the two drugs on microtubule dynamics. In a cell-free MTP-containing model system, plinabulin inhibited microtubule formation more potently than colchicine, as measured by turbidity spectra (Fig. 1A). The concentrations of plinabulin and colchicine at which polymerization was inhibited by 50% (IC₅₀) were 2.4 µM [standard deviation (SD) +/- 0.4, N=4 independent experiments] and 7.6 µM (SD +/- 2.4, N=3), respectively. The tubulin polymerization profiles obtained in the presence of different concentrations of plinabulin or colchicine differed in two ways: First, the initial rate of increase in absorbance over time decreased with increasing drug concentrations of plinabulin, indicating that there is a lag period for microtubule formation. In contrast, the initial rate of polymerization is unchanged at all concentrations of colchicine. Second, microtubule formation in the presence of plinabulin does not reach steady state at high drug concentrations, as indicated by the absorbance values that increase linearly with time, whereas in the presence of high concentrations of colchicine steady state is readily reached. These data are suggestive of a reduction of the pool of soluble, assembly-competent tubulin in the presence of plinabulin. In contrast, colchicine binds nearly irreversibly and induces a rate-limiting conformational alterations in tubulin that, once incorporated into the lattice, inhibits further microtubule polymerization at substoichiometric concentrations (12). Transmission electron microscopy was performed to further validate these results. No microtubule aggregates were identified in the presence of plinabulin 1.25 µM (Fig. 1B) and 2.5 µM (data not shown). The mean microtubule length was however potently reduced to 34% of the DMSO-treatment group at 1.25 µM plinabulin, whereas colchicine 1.25 µM reduced the mean microtubule length to 63% (Fig. 1C; Fig. S1). Next, how these results translated in a cellular system was evaluated. The IC₅₀ of plinabulin for inhibiting mitosis in MCF-7 breast cancer cells was 17 nM and mitosis was halted at the prometaphase (Fig. S2), suggesting that tubulin bound by plinabulin may not be assembly competent to be incorporated into microtubules.

In consideration to the limited activity of chemotherapy in various cancer types with constitutive KRAS activity, whether the distinct mechanism of action of plinabulin yielded antitumor activity in these types of cancer was evaluated. In a panel of 8 human cell lines derived from pancreatic, colorectal and renal cancer, plinabulin inhibited growth in the nanomolar range in all cell lines, except for the pancreatic cancer cell line HPAC (Fig. 2A). This led the present study to also investigate the putative anticancer effects of plinabulin in vivo in a panel of five KRAS mutated xenograft models. In contrast to the in vitro results, plinabulin monotherapy failed to inhibit the growth of LoVo and HCT-15 colorectal cancer (Fig. 2B; Fig. S3A) and exerted no more than a moderate effect on the growth of MDA-MB-231 breast cancer alone (Fig. 2C). The growth of the KRAS mutated DU-145 prostate cancer (Fig. 2D) and A549 lung adenocarcinoma (Fig. S3B) models was not affected by plinabulin monotreatment. However, in combination with standard chemotherapeutic agents administered in these cancers, the additive or synergistic effects of plinabulin were observed on the inhibition of tumor growth, including combinations with the topoisomerase inhibitor irinotecan and with the tubulin targeting agents, paclitaxel and docetaxel (Fig. 2; Fig. S3). These data point towards effects of plinabulin other than direct cytotoxicity and therefore led to down-stream KRAS signaling being evaluated next.

In non-small cell lung and other types of cancer, downstream effects of KRAS signaling include the activation of the MEK/ERK and PI3K/Akt pathways (3). Furthermore, ERK is regulated by a variety of converging pathways that are activated under cellular stress including chemotherapy with tubulin-targeting agents, whereas the key activator of Akt is the KRAS target phosphoinositide 3-kinase (PI3K) (13). The proto-oncogene phosphatase and tensin homolog (PTEN) is the key inhibitor of PI3K, therefore rendering intact PTEN a pre-requisite for KRAS-dependency of cancer cells (14). In PTEN proficient A549 lung cancer cells, disassembly of the microtubule network utilizing plinabulin concentrations in the range of its IC₅₀ for in vitro tubulin depolymerization led to rapid accumulation of KRAS in early endosomes (mean endosomal EEA1-colocalized KRAS in DMSO vs. Plinabulin =2 vs. 31%, P<0.001, N=5) (Fig. 3A and B). Furthermore, overall KRAS levels were decreased upon plinabulin treatment, which may reflect lysosomal degradation, but this needs to be investigated further. The two key down-stream effectors of KRAS signaling, ERK and Akt, were affected differently by plinabulin treatment and subsequent KRAS depletion: ERK phosphorylation was increased, but epidermal growth factor (EGF)-stimulated Ser473-phosphorylation of Akt was reduced (Fig. 3C; 100 ng/ml EGF for indicated times) (15). These findings indicate that displacement of KRAS and potentially other tubulin-dependent kinases may yield decreased downstream PI3K signaling, while alternative signaling pathways act on the ERK signaling cascade.

Next, the specificity of plinabulin effects on KRAS driven types of cancer in a simplified model system that was not biased by co-mutations was determined. For this purpose, two PTEN-proficient glioma gene transfer models that differed solely in the absence or presence of constitutively active KRAS were employed. Gliomas were generated in the brains of Ink4a/Arf-deficient mice expressing the avian t-va receptor under control of the nestin promoter (N/t-va:Ink4a/Arf^-/-) to allow gene transfer utilizing the avian retrovirus RCAS for overexpression of PDGFB and GFP or KRAS^G12A. Although activating KRAS mutations are rare in gliomas, hyperactivation of RAS signaling is a key component of gliomagenesis and histologically, these gliomas resemble the human disease (16). Furthermore, given that brain metastases are an important cause of cancer-associated death (17-19), the present study aimed to evaluate whether plinabulin was capable of penetrating the blood brain barrier to a relevant extent. Penetration of the blood-brain barrier by plinabulin is supported by the observation that in a Quantitative Hole Body Autoradioluminogrphy study, plinabulin rapidly entered the brain parenchyma (Fig. S4). Treatment with plinabulin prolonged survival in KRAS-expressing gliomas (Fig. 3D), but not in tumors that were solely driven by PDGFB and Ink4a/Arf loss (Fig. 3E), therefore confirming penetration of the blood brain barrier to a relevant extent and supporting the notion of efficacy of plinabulin specifically in KRAS-mutated tumors.