STK405759 as a combination therapy with bortezomib or dexamethasone, in in vitro and in vivo multiple myeloma models

Multiple myeloma (MM) remains an incurable hematological malignancy. Combination regimens of conventional and novel drugs have improved patient’s survival. However, most patients inevitably relapse and become refractory to the current therapeutic armamentarium. We investigated the efficacy of combining the microtubule-targeting agent STK405759 with dexamethasone or bortezomib in vitro and in vivo. STK405759 combined with dexamethasone or bortezomib had synergistic cytotoxic activity in RPMIS, CAG and MM1.S human MM cell lines through activation of caspase 2, 3, 8, 9 and PARP. These treatments remained cytotoxic in the presence of bone marrow stroma cells. In other MM cells, including cells resistant to vincristine, melphalan, mitoxantrone or dexamethasone, these combinations decreased significantly survival as compared to single agents. In in vivo studies, STK405759 disrupted existing blood vessels in xenograft tumors, acting not only as a cytotoxic agent but also as an anti-angiogenic drug. Mice treated with STK405759 in combination with dexamethasone or bortezomib resulted in greater tumor growth inhibition, increased overall response and prolonged survival as compared to as compared to BTZ or DEXA alone. Their anticancer activity was mediated by activation of apoptosis and reduction of tumor microvessel density. These preclinical studies provide the rationale for future clinical trials of STK405759, dexamethasone and bortezomib combinations to improve the outcome of multiple myeloma patients.


INTRODUCTION
Multiple myeloma (MM) is a tumor of clonal plasma cells in the bone marrow, often associated with bone lesions, kidney dysfunction, impaired immunity, and anemia. Multiple myeloma accounts for 1.5% of all cancers and approximately 13% of all hematologic malignancies [1].
The introduction of novel drugs, including immunomodulatory (lenalidomide, pomalidomide), proteasome inhibitors (bortezomib [BTZ], carfilzomib, ixazomib), and more recently, the deacetylase inhibitor panobinostat and the monoclonal antibodies daratumumab and elotuzumab, have improved outcomes [2][3][4]. However, most patients eventually relapse and become resistant to current therapies. The prognosis of MM patients who have received at least three prior lines of therapy is very poor, with event-free survival and overall survival of only 5 and 13 months, respectively [5,6]. Hence, the pursuit for STK405759 as a combination therapy with bortezomib or dexamethasone, in in vitro and in vivo multiple myeloma models newer classes of drugs and novel therapeutic approaches is critical, especially in the high-risk and relapsed-refractory settings.
The use of microtubule-targeting agents (MTAs), has significantly increased response and survival among patients with many cancers including solid tumors and hematological malignancies [7][8][9]. The tubulin depolarizing agent vincristine has been used largely in different combination regimens for refractory MM [10]. It is effective until patients develop drug resistance or side-effects related to neurological toxicity [11][12][13][14]. Therefore, developing MTAs that are less toxic, more potent and can overcome drug resistance, represents a promising strategy for MM therapy.
Recently, we showed that STK405759 is a tubulin depolymerizing agent with potent, selective cytotoxic activity against myeloma cells. STK405759 as a single agent, reduced tumor burden and increased survival of treated mice in a MM xenograft model [15].
In the present study, we investigated the efficacy and toxicity of STK405759 in combination with dexamethasone (DEXA) or with the proteasome inhibitor BTZ in resistant MM cell lines and in a xenograft mice model.

STK405759 in combination with DEXA or BTZ reduces viability of MM cells
Previously, we showed in vitro synergistic cytotoxic activity between STK405759 and DEXA or BTZ in human RPMI-S and MM.1S cell lines [15]. Here, we extended their effect to a larger spectrum of MM cells.
STK405759 exposure in combination with DEXA or BTZ significantly reduced survival of CAG, JJN3, OcyM5, OPM2 and KMS-11 MM cells, as compared to single drugs, even when cells were minimally responsive to the single agent. However, this effect was synergistic only in CAG cells (CI: 0.9). Importantly, low concentrations of STK405759 combined with DEXA or BTZ treatments of the melphalan (RPMI-LR5) and vincristine resistant (RPMI-DOX40) cell lines showed enhanced cell death as compared to each drug alone. In contrast, when treated with combined DEXA or BTZ and STK405759, cells resistant to DEXA (MM1.R) and mitoxantrone, (RPMI-MR20) showed a cytotoxic response similar to STK405759 treatment alone ( Figures  1-2). The triple combination of STK405759 with DEXA and BTZ didn't changed the cytotoxicity as compared to STK405759-DEXA, STK405759-BTZ or DEXA-BTZ treatments in RPMI-S, MM1.S and CAG cells.

STK405759 in combination with DEXA or BTZ overcomes bone marrow resistance
The MM-host bone marrow microenvironment confers growth, survival and drug resistance to MM cells [16]. RPMI-S, MM.1S and CAG MM cells were stained with CFSE, co-cultured with HS5 bone marrow stroma cells, and treated for 48 h with the different drugs and their combinations. Cell viability was then analyzed with propidium iodide. HS5 cells decreased the cytotoxicity of DEXA and BTZ on RPMI-S and MM.1S cells, but not of STK405759. CAG co-cultured with HS5 cells were sensitive to BTZ and STK405759, but not to DEXA. The combination of STK405759 with DEXA or BTZ increased their cytotoxicity on MM co-cultured cells, as compared with STK405759 alone. HS5 viability was not affected by any treatment in co-cultured cells (Figure 3).

Cytotoxic mechanisms mediating the anti-MM activity of STK405759 plus DEXA or BTZ
In order to elucidate the cellular mechanisms driving the advantage of combined therapy observed on viability, we tested the expression level of proteins related to cell death. Three MM cell lines (RPMI-S, MM.1S and CAG) were treated with STK405759 alone or in combination with DEXA or BTZ for 24 h and their proteins isolated and immunoblotted. The pattern of cell death pathway activation differed among the cell lines after STK405759, DEXA or BTZ treatment. Thus, STK405759 increased cleavage of caspase 3 in MM.1S and CAG, but not in RPMI-S cells, whereas caspase 2 activation and decreased myeloid cell leukemia-1 (mcl-1) expression levels occurred mainly in RPMI-S cells. Poly-(ADP-ribose) polymerase (PARP) and caspase 8 activation were common to all tested cells after STK405759 treatment. DEXA treatment activated PARP and microtubule-associated protein light chain 3 (LC3) in CAG cells, whereas BTZ activated caspase 8 on MM.1S cells, caspase 2 on CAG cells, and PARP on RPMIS, MM.1S and CAG cells. The combined treatments included activation of each of the proteins activated by the single agents alone, confirming the enhanced cytotoxicity of these combinations in cell viability assays ( Figure 4A).
We showed that STK405759 inhibited tubulin polymerization in a concentration-dependent manner in an in vitro cell-free system and in MM cells [15].
Here we observed that neither DEXA nor BTZ affected the level of soluble or polymerized tubulin in RPMI-S cells. The combination of each of them with STK405759, resulted in reduced expression of polymerized tubulin ( Figure 4B-4C).

STK405759 combined with DEXA or BTZ exhibited significantly enhanced antimyeloma efficacy in vivo
Based on the efficacy of STK405759 combined with DEXA or BTZ in targeting MM cells in vitro, we validated these findings in vivo. Mice with a subcutaneous human www.oncotarget.com Combination index (CI) was calculated using compusyn software. Each treatment was performed in triplicate in three independent experiments and presented as mean ± SE. Values were normalized to the drug-free control. Results are presented as mean ± SE. * p < 0.05; # p < 0.0001 t-test. www.oncotarget.com MM tumor were treated with STK405759, DEXA, BTZ, STK405759+DEXA or STK405759+BTZ for 3 months.
As a single agent, DEXA was more potent than was STK405759 (p < 0.0001). This difference decreased from day 80 (p = 0.2) of treatment due to development of resistance in the DEXA treated group. Importantly, treatment with STK405759 and DEXA was significantly more effective than with DEXA alone (p = 0.002 on day 31 and p = 0.000001 on day 80, t-test), with reduced tumor burden in all the treated mice. The combination induced a clear inhibition of tumor growth, which was maintained at least for 90 days. On day 49, the tumors from DEXA treated mice relapsed and started to grow, reaching a tumor volume 5 times larger than that of the Results are presented as mean ± SE. * p < 0.05; # p < 0.0001 t-test. www.oncotarget.com STK405759 + DEXA treated mice on day 80. Complete tumor regression was observed in 50% of the mice treated with STK405759 plus DEXA and remained stable for 60 days after the last treatment.
The improved efficacy of STK405759 combined with DEXA was confirmed by the weight of the extracted tumors at the end of the experiment (p = 0.007). DEXA plus STK405759 significantly improved survival relative to the vehicle-treated group (p < 0.0001) ( Figure 5).
In vivo, the efficacy of BTZ and STK405759 as single drugs were similar (p > 0.5, t-test).
Combined BTZ and STK405759 treatment significantly reduced tumor size as compared to each component administered alone (p < 0.5, t-test). After 42 days of treatment with BTZ, STK405759 or BTZ + STK405759, tumor volume decreased ~60%, ~50% and ~80%, respectively ( Figure  6). The improved efficacy of STK405759 when combined with BTZ was also confirmed by the weight of the tumors extracted after the final tumor volume measurement (p < 0.05 t-test; Figure 6). With respect to toxicity, decrease in body weight was not observed in mice receiving combined treatments. STK405759 resulted in better survival than BTZ-treated mice (p = 0.036, chi-square). STK405759 combined with BTZ improved survival significantly, as compared to vehicle (p = 0.003, chi-square) and to a lesser extent in the groups treated with BTZ or STK405759 alone, respectively ( Figure 6).
Importantly, blood tests revealed no toxicity-related complications with either combination tested. There were no signs of anemia, as evidenced by normal red blood cell, hemoglobin, hematocrit and platelet values relative to naïve mice, or of hepatic or renal failure ( Supplementary  Figures 1, 2), suggesting a favorable therapeutic index for these regimens.

STK405759 has antiangiogenic activity and induces apoptosis in vivo
Tumor samples were analyzed with H&E staining to evaluate morphological changes and by TdT-mediated dUTP nick-end labeling (TUNEL) assays to evaluate apoptosis. Anti-CD34 monoclonal antibody was used to estimate microvessel density (MVD) in RPMI-S cells, in vivo. Tumors from control mice had typical histologic appearance, whereas those from mice treated with the drug combinations had lower MM cell density, as well as signs of necrosis.
The combination of STK405759 with DEXA or BTZ increased the number of necrotic areas and of TUNEL-positive apoptotic tumor cells as compared to treatment with vehicle or either agent alone (Figure 7), confirming the activation of apoptosis observed in the in vitro results, discussed earlier.
STK405759 significantly decreased MVD after therapy in treated mice, supporting an antiangiogenic effect of STK405759 in vivo. The efficacy of STK405759 plus DEXA or BTZ was also enhanced by reduced vascular areas and fewer branched vessels in tumor tissues (Figure 7), with reduced expression level of CD34 endothelial stem cell markers.

DISCUSSION
MM remains incurable in most patients, prompting an ongoing search for additional therapeutic options, including multidrug-combinations that induce long-term tumor regression with little cross-resistance and low toxicity.
Here, we showed that the combination of STK405759 and DEXA or BTZ increased cytotoxicity, as compared to each drug alone, through activation of each of the apoptotic-related proteins.
In an in vivo xenograft model, treatment with STK405759 combined with DEXA or BTZ resulted in significant inhibition of tumor growth, sustained tumor regression and increased overall response compared to treatment with each drug alone.
Bone marrow angiogenesis correlates with disease progression and prognosis in MM patients [17,18]. The grade of vascularization before initiation of therapy was found to be a predictive factor for survival [19]. BTZ exhibits anti-angiogenic activity both in vitro and in vivo [20,21] and MM patients treated with BTZ show a significant decrease in MVD [22]. Similarly, MTAs demonstrate antiangiogenic activity in a wide range of malignancies [23,24]. In the current study, STK405759 disrupted existing blood vessels in xenograft tumors, acting both as a cytotoxic agent and as an anti-angiogenic drug. The anti-angiogenic effect of STK405759 might contribute to the increase efficacy observed after its combination with DEXA and BTZ in vivo.
Historically, vincristine has been used as part of conventional chemotherapeutic regimens for MM, as patients were treated with the combination of vincristine, doxorubicin and DEXA (VAD) [10,[25][26][27][28]. This combination therapy has been replaced in the last decade due to hematological (granulocytopenia and thrombocytopenia) and non-hematological toxicity (neurotoxicity and impaired cardiac function), modest activity, the development of chemoresistance and the emergence of novel therapeutic agents. The main mechanism of vincristine chemoresistance was through overexpression of the multidrug efflux pumps P-glycoprotein [14,29,30]. We found that the combination of STK405759 with DEXA or BTZ was not affected by this mechanism of resistance. In addition, blood tests of treated mice showed no sign of hematological toxicity.
Various groups reported that the use of vincristine combined with anthracyclines significantly reduced the antitumor effects, as compared with the administration of each drug alone [31][32][33]. In accordance with these results, we also found antagonistic activity between STK405759 and doxorubicin [15]. The antagonistic effect between MTAs and anthracyclines might explain the modest activity of historical combination therapy (e.g. VAD). Despite this antagonistic effect, a recent case report described a patient with secondary plasma cell leukemia refractory to BTZ-DEXA and to lenalidomide-DEXA therapies, who achieved partial remission after vincristine-doxorubicin-DEXA treatment, suggesting potential benefits of using MTA agents in the treatment of unresponsive patients [34].
In summary, we showed that the use of STK405759 with BTZ or DEXA significantly enhanced efficacy and increased survival in a MM mice model, as compared to each drug alone, and without observable side effects. These data provide a rationale for further clinical evaluation of STK405759in combination with DEXA or BTZ for patients with relapsing and refractory MM.
Evaluation of efficacy included inhibition of tumor growth, survival, blood tests, animals' vital signs and gross pathology. Tumor size was measured by caliper measurements of the longest perpendicular tumor diameters, every 3-4 days to estimate tumor volume, using the formula representing the 3-dimensional volume of an ellipse: 4/3× (width/2) exp(2× (length/2)). Blood was drawn on days 20 and 44 of treatment. The blood tests were performed by Tel Hashomer Core Equipment Facilities. Animals were sacrificed when the tumor reached 2.5 cm, or ≥20% decrease in body weight or appeared moribund, to prevent unnecessary morbidity to the mice. Overall survival, was defined as time between initiation of treatment and sacrifice or death. The Kaplan-Meier end-product analysis was used to compare control vs. each group of treated mice.

Histochemistry
The tumors were fixed in 4% paraformaldehyde for 24 h (Sigma-Aldrich, St. Louis, MO, USA), washed with PBS, dehydrated in increasing alcohol concentrations and embedded in paraffin blocks. Sections were deparaffinized and rehydrated, treated with proteinase K (20 μg/ml) for 15 min and washed in PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 min. Fragmented nuclear DNA, associated with apoptosis in histological sections, was labeled in situ with digoxigenin-deoxyuridine (dUTP), introduced by terminal deoxynucleotidyl transferase (TdT), using ApopTag ® peroxidase in situ apoptosis detection kit according to manufacturer's instructions (Intergen, Oxford, England, UK). The reaction was terminated using the ApopTag ® stop buffer followed by anti-digoxigenin-peroxidase application and the labeled nuclei were detected with ACE substrate as the chromogen (Sigma-Aldrich, St. Louis, MO, USA). MVD were labeled using anti-CD34 antibody (Abcam, ab81289, USA). The slides were examined using a Zeiss microscope, and images were processed using ZEN digital imaging software. www.oncotarget.com

Statistical analysis
The differences in drug-treated vs. control cultures was determined using Student's t-test. Data are presented as mean ± standard error (SE). For in vivo experiments, survival was assessed using Kaplan-Meier curves and Chisquare analyses.

Author contributions
Gabriela Rozic: conception and design of the study, acquisition of data, analysis and interpretation of data; drafting of the article. Lena Paukov: acquisition of data. Ziv Cohen: acquisition of data. Irit Shapira: acquisition of data. Adrian Duek: revising the article for important content. Ohad Bejamini: revising the article for important content. Abraham Avigdor: revising the article for important content. Arnon Nagler: revising the article for important content. Igor Koman: analysis and interpretation of data, revising the article for important content. Merav Leiba: conception of the study, analysis and interpretation of data; final approval of the version to be published.