Exploiting Protein Translation Dependence in Multiple Myeloma with Omacetaxine-based Therapy

Abstract

Purpose:

The prognosis of patients with multiple myeloma (MM) who are resistant to proteasome inhibitors, immunomodulatory drugs (IMiDs), and daratumumab is extremely poor. Even B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cell therapies provide only a temporary benefit before patients succumb to their disease. In this report, we interrogate the unique sensitivity of MM cells to the alternative strategy of blocking protein translation with omacetaxine.

Experimental Design:

We determined protein translation levels (n = 17) and sensitivity to omacetaxine (n = 51) of primary MM patient samples. Synergy was evaluated between omacetaxine and IMiDs in vitro, ex vivo, and in vivo. Underlying mechanism was investigated via proteomic analysis.

Results:

Almost universally, primary patient MM cells exhibit >2.5-fold increased rates of protein translation compared to normal marrow cells. Ex vivo treatment with omacetaxine resulted in >50% reduction in viable MM cells. In this cohort, high levels of translation serve as a biomarker for patient MM cell sensitivity to omacetaxine. Unexpectedly, omacetaxine demonstrated synergy with IMiDs in MM cell lines in vitro. In addition, in an IMiD-resistant relapsed patient sample, omacetaxine/IMiD combination treatment re-sensitized the MM cells to the IMiD. Proteomic analysis found that the omacetaxine/IMiD combination treatment produced a double-hit on the IRF4/c-MYC pathway, which is critical to MM survival.

Conclusion:

Overall, protein translation inhibitors represent a potential new drug class for myeloma treatment and provide a rationale for conducting clinical trials with omacetaxine alone and in combination with IMiDs for patients with relapsed/refractory MM.

INTRODUCTION

Multiple myeloma (MM) is an aggressive hematologic malignancy characterized by over-proliferation and tissue invasion by malignant plasma cells that retain the fundamental biologic attributes of antibody production and secretion. MM afflicts more than 30,000 Americans with increasing prevelance.¹ In the early 21^st century, with the introduction of proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs) and monoclonal antibodies, the average life expectancy of MM patients has drastically improved.² Beginning in 2015, the anti-CD38 monoclonal antibody daratumumab emerged as another important treatment option.³ However, MM remains incurable, and drug resistance to all of the above agents is referred to as “penta-refractory,” with a median survival of approximately nine months.⁴ Throughout the disease course, patients are debilitated by bone tumors, pathologic fractures, kidney failure, and immunosuppression, eventually becoming fatal. Thus, new therapies that retain their activity in advanced MM patients who have developed IMiD, PI, and daratumumab resistance are urgently needed.

We are focused on the development of new drugs for penta-refractory MM, and became interested in omacetaxine as a drug with therapeutic potential. Omacetaxine is FDA-approved for chronic myeloid leukemia (CML) and acts via a unique mechanism among anti-cancer drugs by binding to the A-site cleft of ribosomes and blocking the initial elongation step of protein synthesis.⁵ In CML, translation inhibition may act by downregulating key metastable oncoproteins such as the anti-apoptotic protein MCL1 and the oncogenic transcription factor c-MYC.^6-8 Both MCL1 and c-MYC are known to be important pro-survival proteins frequently overexpressed and activated in MM.^9,10 Accordingly, most MM cell lines are dependent on MCL1 and c-MYC.^11,12 Notably, genomic abnormalities involving the c-MYC locus occur in a subset of MM patients and upregulate its expression.^13-15 In addition, genomic amplification of the MCL1 gene, which resides on chromosome 1q, occurs frequently in multiple myeloma, including ~80% of patients with relapsed MM.^16-18 Thus, MCL1 and c-MYC are attractive targets in MM.

Considering the above evidence, its stands to reason that omacetaxine may be effective in targeting MM. Work from multiple reports has demonstrated that omacetaxine has potent cytotoxicity against MM cell lines in vitro and in vivo.^19-22 Further supporting this approach, inhibiting the initiation step of protein translation by knocking down eIF4E hinders MM growth and survival.^24,25 In addition, Manier and colleagues demonstrated high rates of protein translation related to c-MYC activity in MM and identified the translational initiation inhibiting rocaglates to be detrimental to MM survival.²³ MM cells are generally sensitive to agents that disrupt their fragile state of proteostasis, including PIs which have a broad effect on protein degradation, and IMiDs, which directly cause the degradation of Ikaros proteins, leading to decreased levels of c-MYC and IRF4 and downregulation of their downstream genetic programs.^26-28 Protein translation inhibition is also known to decrease the levels of short half-life proteins such as c-MYC, MCL1 and other prosurvival proteins in MM.^20,21,23 Thus, multiple groups have supported that protein translation inhibition has the potential to be an effective strategy to target MM cells.

Here, we report further preclinical assessments of omacetaxine with a focus on primary samples from patients with multiple myeloma. We first analyzed the anti-myeloma activity of omacetaxine and the MM cell protein translation rates and in samples from a cohort of MM patients ranging from newly diagnosed to having advanced disease. In doing so, we found that high rates of protein translation in MM cells served as a biomarker for potent anti-myeloma cytotoxicity of omacetaxine treatment. In seeking drug combination partners, we establish the IMiDs lenalidomide and pomalidomide were synergistic with omacetaxine in vitro, in vivo, and ex vivo. In evaluating the mechanism for that synergy, we found a double-hit on the key MM cell survival factors IRF4 and c-MYC. Overall, omacetaxine exhibits potent and selective anti-myeloma activity that is retained in PI/IMiD refractory disease, and in combination with IMiDs represents a synergistic regimen to test in patients.

MATERIALS AND METHODS

Drugs

Omacetaxine mepesuccinate/homoharringtonine (referred to herein as omacetaxine or Oma) and (+)-JQ1 were purchased from Selleckchem. The PIs, IMiDs, and dexamethasone were purchased from Thermo Fisher Scientific (Waltham, MA). Four-Hydroperoxy Cyclophosphamide (4-HC) was purchased from Santa Cruz Biotechnology (Dallas, TX), and Dara was obtained from the University of Colorado Health Pharmacy.

Cell lines

MM cell lines RPMI-8226, U266, NCI-H929, OPM-2, AMO-1, MM.1S, and MM.1R, were obtained from the American Type Culture Collection (ATCC), and validated via short tandem repeat polymorphism (STR) profile match analysis. Cell lines were cultured at 37°C with 5% CO₂ in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific, Asheville, NC).

Cell Line Cytoxicity

Cell proliferation was determined using the CellTiter-Glo Luminescent Cell Proliferation assay (Promega, Madison WI) per manufacturer instructions following drug incubations in 96-well culture plates at 37°C for 96 h. Results were obtained using a Vmax Kinetic Microplate Reader (Molecular Devices, Sunnyvale CA). Cell viability was also measured via flow cytometry using the LIVE/DEAD Fixable Near-IR Dead Cell Stain (Invitrogen, Carlsbad CA).

Drug Combination Studies

Drug combination studies were set up using a full matrix design with myeloma cell lines or primary samples. Synergy was calculated using ZIP delta score via the SynergyFinder online tool (https://synergyfinder.fimm.fi).²⁹

Patient Sample Processing

Bone marrow aspirates from patients with MM or smoldering myeloma were obtained and mononuclear cells (MNCs) were isolated from the samples using SepMate Ficoll-Plaque tubes (StemCell Technologies) according to the manufacturer’s instructions. CD138-positive cell selection was not performed for any of the experiments. Samples were cryopreserved in freezing medium consisting of Iscove’s Modified Dulbecco’s Medium (IMDM), 45% fetal bovine (FBS), and 10% dimethylsulfoxide (DMSO) at 10 million cells/mL.

Ex Vivo Drug Sensitivity Testing

Patient MM cell drug sensitivity testing was performed with our My-DST platform as recently described.³⁰ In brief, de-identified primary myeloma mononuclear cells were cultured in RPMI1640 medium containing L-glutamine with 10% FBS and 100 U/mL penicillin, 100 ug/mL streptomycin (Thermo Fisher Scientific), and 2 ng/mL interleukin 6 IL-6 (PreproTech) at 37°C. Cells were then transferred to 96-well plates at 4.5 x 10⁵ cells/mL (90,000 MNCs per well) and treated for 48 h.

Flow Cytometry

After 48 h treatment, cells were washed in 1X cold DPBS and re-suspended in BD Brilliant Stain Buffer (BD Biosciences, San Jose CA) in a 96-well V-bottom plate. Cells were treated with FcR Blocking Reagent (Miltenyi Biotec, San Diego CA) for 5 min and then surface stained with anti-CD19-BV786 (SJ25C1), anti-CD45-BV510 (HI30), multi-epitope anti-CD38-FITC (American Laboratory Products Company), anti-CD138-BV421 (MI15), and anti-CD319-Alexa647 (235614) or anti-CD46-Alexa647 (E4.3) antibodies for 10 min on ice. Intracellular staining with anti-kappa-BV605 and anti-lambda-PE light chains was performed after paraformaldehyde fixation and permeabilization. All flow cytometry antibodies were purchased from BD Biosciences except where noted. After staining, samples were washed and resuspended with 100 μl DPBS containing 2% FBS (FACS buffer). Viability was determined with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen, Carlsbad CA) at 1:10,000. Flow cytometry data was collected using a BD FACSCelesta Multicolor Cell Analyzer (BD Biosciences, San Jose CA) equipped with a high throughput sampler (HTS). Analysis was completed using FlowJo software (FlowJo LLC, Ashland OR). Myeloma populations were analyzed gating for CD19, CD45, CD38 and CD138 based on the cells expressing clonal light chain restriction. Intracellular flow was used to measure MCL1 (22/Mcl-1 antibody, BD Biosciences), c-MYC (Y69 antibody Alexa-488, Abcam), IRF4 (3E4 antibody, eFluor710, In Vitrogen) and BCL2 (BMS1028 antibody, Thermo Fisher) expression. Intracellular staining of MCL1 and BCL2 was conducted following permeabilization with the Cytofix/Cytoperm kit (BD).

Protein Translation Measurement

Protein synthesis levels were determined in vitro utilizing the puromycin analog O-propargyl-puromycin (OP-Puro) in the protein synthesis assay kit (Cayman Chemical) according to manufacturer instructions. For in vivo protein synthesis, engrafted NSG mice were injected IP with 500 μg puromycin (Gibco), then euthanized after 1.5h, marrow was flushed, and cells stained with anti-puromycin antibody (12D10, Alexa-488, Millipore Sigma), followed by flow cytometry.

BH3 Priming Assay

To evaluate BCL2 family member dependence in myeloma cell lines and patient samples, we adapted the published assay for BH3 profiling.³¹ Following surface staining, cells were exposed to the various BH3 mimetic peptides to test for BH3 priming, including BIM, BAD, and NOXA. Mitochondria were permeabilized with digitonin, and cells were fixed with paraformaldehyde. BH3 profile was determined using flow cytometry and staining for cytochrome-c loss.

Myeloma Cell Line Xenografts

For in vivo assessment of omacetaxine, 5 x 10⁵ MM.1S cells stably expressing firefly luciferase were injected intravenously into NSG mice (Jackson Labs, Bar Harbor, ME) to generate an orthometastatic MM xenograft model, as done previously.³² When untreated, the NSG mice succumb to orthometastatic myeloma-like disease ~50 days after implantation in this model. Bioluminescence imaging (BLI) was used to monitor graft status weekly and mice were stratified into treatment groups to normalize BLI at start of study. Treatment was administered by intraperitoneal (IP) injection in a final volume 0.5 ml of PBS with 0.5% FBS. Tumor status was assessed by BLI, and the results were analyzed by Living Image (PerkinElmer).

Myeloma Cell Metabolism

MM cell lines were incubated with pomalidomide for 24 h, with omacetaxine for 4 h, or in staggered combination. The cells were washed and resuspended in Agilent Seahorse XF DMEM Medium, pH 7.4 (Agilent Technologies, Santa Clara, CA). Glycolytic flux and the extracellular acidification rate (ECAR) were measured on a Seahorse XFe96 Analyzer using the Seahorse XF Glycolysis Stress Test Kit according to manufacturer instructions (Agilent Technologies).

Myeloma Cell Proteomics Analysis

Cells were lysed with RIPA buffer (Thermo Fisher Scientific) and digested according to the FASP (Filter Aided Sample Preparation) protocol.³³ Recovered peptides were dried, desalted, and concentrated on Thermo Scientific Pierce C18 Tips. Cell lysates were analyzed using an Orbitrap Fusion mass spectrometer connected to an EASY-nLC 1200 system (Thermo Fisher Scientific) with a nanoelectrospray ion source. Data was acquired using the Xcalibur™ (version 4.1) software. MS/MS spectra were extracted, and Proteome Discoverer Software (ver. 2.1.0.62) was used to convert the raw data into mgf files. The files were then searched against a human database using an in-house Mascot server (Version 2.6, Matrix Science). Scaffold (version 4.8, Proteome Software, Portland, OR) was used to validate MS/MS based peptide and protein identifications. Protein identifications were established using >99.0% probability cutoff and contained at least two identified unique peptides. Heatmaps, principal component analysis (PCA) and volcano plots were generated using MetaboAnalyst software (McGill University, Montreal, Quebec, Canada) and R Studio. Western blots to measure specific proteins were performed with anti-c-MYC (9402 antibody, Cell Signaling Technology), anti-IRF4 (IRF4.3E4, Biolegend), anti-IKZF1 (E-2, Santa Cruz Biotechnology) and anti-IKZF3 (NBP2-24495, Novus Biologicals), with anti-ACTB (C4, Santa Cruz Biotechnology) as a loading control. For western blotting, cells were lysed with RIPA buffer (Thermo Fisher Scientific). Protein concentrations were determined with the DC protein assay (BioRad). Equal amounts of protein were loaded and resolved by SDS-PAGE electrophoresis and transferred to PVDF membranes for blotting. Antibodies were incubated overnight at 4°C in 5% BSA TBS (Boston BioProducts). Membranes were incubated with secondary antibody for two hours.

Statistics

Statistics and figures were generated using Prism 6 software (GraphPad Software, San Diego, CA). All data are presented as mean and standard deviation. EC₅₀ values were determined by non-linear regression variable slope inhibitor vs response curves. Two-tailed Student’s t test was used for comparing two means. When comparing more than two means, ANOVA was used with Tukey’s correction. Survival analyses were conducted using SAS version 9.4 (SAS Institute) with Cox proportional hazard models to calculate hazard ratios (HRs). Levels of statistical significance are shown by: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Study Approval

Bone marrow aspirates were collected from patients at the University of Colorado Blood Cancer and Bone Marrow Transplant Program after written informed consent and in accordance with the Declaration of Helsinki. Samples from patients with MM or smoldering myeloma were obtained from the hematologic malignancies tissue bank with protocol approval from the Colorado Multiple Institutional Review Board. All animal studies were conducted in compliance with protocols reviewed and approved by the University of Colorado Institutional Animal Care and Use Committee.

RESULTS

Omacetaxine Has Broad Anti-Myeloma Efficacy Against Myeloma Cells

We tested omacetaxine in MM cell lines in vitro and in MM patient bone marrow aspirates ex vivo. First, we verified the reported cytotoxicity of omacetaxine in cell culture using U266, H929, MM.1S, MM.1R, and RPMI-8226 MM cell lines.^20,21 Omacetaxine inhibited cell proliferation with an EC₅₀ range of 6-28 nM after a 96 h incubation (Figure 1A). In a timecourse study, omacetaxine-mediated induction of apoptosis started at 2 h, and cell death began after 24 h (Figure 1B-C, Supplementary Figure 1A). As further evidence of its rapid and drastic effects, omacetaxine treatment reduced glycolytic and oxidative phosphorylation metabolism in MM cell lines after 4 h, as measured using the Seahorse assay (Figure 1D, Supplementary Figure 1B-C). Thus, omacetaxine rapidly reduced metabolism, and induced apoptosis and cell death in all MM cell lines tested.

Figure 1. Omacetaxine cytotoxicity in myeloma cell lines and primary patient samples.

(A) Non-linear regressions analysis of cell proliferation assay results for five MM cell lines treated with increasing doses of omacetaxine (Oma) for 96 h. (B) Co-staining with Annexin V and DAPI of the MM.1S cell line treated with 50 nM omacetaxine for 48 h. (C) Timecourse of the induction of apoptosis with omacetaxine (50 nM) treatment in MM.1S cells. (D) Omacetaxine treatment reduces myeloma cell line metabolism, as measured by ECAR after a 4 h incubation. (E) Flow cytometry gating strategy after ex vivo treatment of primary MM cells from patient HTB-1580 with 50 nM omacetaxine for 48 h. Live cells were gated followed typically by CD45dim-/CD19-(not shown) and finally CD38+/CD138+. (F) Dose response curves for six different MM patient primary samples treated with increasing concentrations of omacetaxine for 48 h show a decline in viable MM cells as measured by multicolor flow cytometry and graphed as % normalized (Norm) to untreated controls. (G) Waterfall plot showing the ex vivo effect of 50 nM omacetaxine treatment for 48 h in 51 patient samples categorized based on their PI and IMiD resistance as measured by Myeloma-Drug Sensitivity Testing.³⁰ Data represent means ± SD, comparisons by two-tailed Student’s t test, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To evaluate the extent to which the in vitro activity of omacetaxine in MM cell lines may be clinically significant, we evaluated omacetaxine activity in primary samples from six different MM patients. The effect of omacetaxine in primary samples was tested by incubating mononuclear cells (MNCs) from patient bone marrow aspirates (n = 6) for 48 h and measuring the number of surviving MM cells by multiparameter flow cytometry, as described.³⁰ With this assay, we found that omacetaxine specifically decreases MM cell viability ex vivo with an EC₅₀ range of 25-225 nM (Figure 1E-F). Using a single concentration to screen additional primary samples, we found that ex vivo treatment with 50 nM omacetaxine for 48 h resulted in a >50% reduction in viable MM cells in 39/50 (78%) patient samples (Figure 1G). Importantly, patients with relapsed/refractory myeloma resistance to PI and IMiDs did not show cross-resistance to omacetaxine, which retained highly potent and specific anti-myeloma activity in single- and double-class refractory patients (Figure 1G). On the other hand, sensitivity to omacetaxine amongst these samples did not correlate with other patient characteristics, including the level of disease involvement in the bone marrow, cytogenetics, and prior lines of therapy (Supplementary Figure 2A-D, Supplementary Table 1). In regards to the effects on normal cells, we have previously found that omacetaxine does not affect the viability of against normal hematopoietic stem cells at concentrations tested up to 100 nM.³⁴ Overall, these findings show that omacetaxine has anti-myeloma activity in samples from a broad range of patients with multiple myeloma, importantly including those with multi-drug resistant disease, at doses that don’t affect normal hematopoietic stem cells.

Omacetaxine Inhibits Highly Active Protein Translation in Myeloma Cells

Since the anti-myeloma effect of omacetaxine was present in a broad range of patient samples, we hypothesized that myeloma cells may require inherently high levels of protein synthesis. We used the O-Propargyl-puromycin (OP-Puro) assay to measure the rate of amino acid incorporation into translating ribosomes by flow cytometry. Primary MM cells exhibited significantly higher levels of baseline protein translation compared to the other non-malignant bone marrow MNCs, with a mean 3.9-fold increased OP-puro signal (n = 17, P = <0.001) (Figure 2A-B). Omacetaxine treatment for inhibited protein translation with an EC₅₀ of 9-12 nM and within 30 mins of dosing, preceding the induction of apoptosis (Figure 2C, Supplementary Figure 3A). Omacetaxine inhibited translation across myeloma cell lines, while bortezomib treatment did not (Supplementary Figure 3B-C). In 14 primary samples tested, translation was significantly reduced after 2.5 h of 50 nM omacetaxine treatment to near normal MNC levels (Figure 2D). Strikingly, using a cutoff of the bottom quartile of baseline MM translation compared to MNCs (2.5-fold or higher), “High Translation” was associated with sensitivity to omacetaxine, and “Low Translation” was associated with relative resistance to omacetaxine (P = 0.0018) (Figure 2E). Myeloma cell protein translation levels were not associated with the patient’s sensitivity to PIs or IMiDs (Supplementary Figure 3D). The high level of translation was also observed in bone marrow plasma cells from healthy donors, and omacetaxine was also cytotoxic to these normal plasma cells (Supplementary Figure 3E-F). Thus, protein translation inhibition is consistent across myeloma cells at concentrations that are also cytotoxic, and the translation rates may serve as a biomarker for response.

Figure 2. Omacetaxine inhibits translation.

(A) Translation levels in primary MM cells and the non-malignant bone marrow mononuclear cells (MNCs) from 17 patients measured using OP-Puro and flow cytometry. (B) Paired T-test of translation level median fluorescence intensity (MFI) in primary MM cells compared to normal MNCs . (C) There is a dose-dependent decrease in translation levels with increasing omacetaxine (Oma) concentrations in the H929 MM cell line and in two primary MM cell samples. (D) Omacetaxine (50 nM) inhibits translation in primary MM cells after 2.5 h. (E) Ex vivo sensitivity of primary sample MM cells to omacetaxine (graphed by % normalized to untreated controls) categorized as high translation vs. low translation using a cutoff of 2.5-fold or greater translation compared to nonplasma cells. (F) MCL1 antigen density on primary MM cells compared to nonplasma cells. (G) MCL1 MFI of six MM cell lines at baseline and after 2.5 h of 50 nM omacetaxine treatment. (H) c-MYC MFI of MM.1S cells after 4 h of 50 nM omacetaxine treatment. Data represent means ± SD, comparisons by two-tailed Student’s t test, **p < 0.01, ***p < 0.001, ****p < 0.0001.

By blocking protein translation, omacetaxine is known to downregulate the expression of short-lived oncoproteins, such as MCL1, in multiple cell types.^6-8,20 Thus, we investigated omacetaxine-induced downregulation of proteins known to be important for MM cell survival, such as MCL1 and c-MYC. Indeed, by quantitative flow cytometry, we confirmed that MCL1 is overexpressed in MM cells and that omacetaxine rapidly downregulates MCL1, but not BCL2, expression in myeloma cells after 2.5 h incubation (Figure 2F-G, Supplementary Figure 4A-B). By comparison, the MCL1 inhibitor S63 was less cytotoxic than omacetaxine (Supplementary Figure 4C)³⁵. To determine whether MCL1 dependence may predict ex vivo response to omacetaxine, we established the BH3 profiling for BCL2 family member priming, as demonstrated in Supplementary Figure 4D with H929 cells known to be MCL1-primed.^11,31 In a subset of our patient sample cohort, we found that omacetaxine exhibited potent cytotoxicity against both MM cells that were primed and MM cells that were not primed for MCL1-mediated apoptosis (Supplementary Figure 4E). Whereas S63 cytotoxicity correlated well with MCL1 priming, omacetaxine demonstrated potent anti-myeloma activity in samples that were not MCL1 primed (Supplementary Figure 4F-G). In addition to MCL1, we also demonstrated that omacetaxine rapidly downregulates c-MYC after 4 h of treatment in MM.1S cells (Figure 2H). Thus, downregulation of MCL1, though important in myeloma cell survival, did not appear to fully explain the spectrum of omacetaxine activity against the disease.

Omacetaxine is Synergistic with Immunomodulatory Drugs

The benefits of combination therapy in MM have been consistently demonstrated in clinical trials and in practice since the early 2000s. As a result, we sought to identify the best clinically available agents to combine with omacetaxine. Using MM cell lines Amo-1, L363, and U266, we combined omacetaxine with various anti-myeloma agents in two-drug combination matrices and measured viability after a 96 h incubation. Omacetaxine combined with lenalidomide or with pomalidomide (Pom) stood out as synergistic in all cell lines tested (Figure 3A, 3C). The δ-score for synergy of omacetaxine with Len and Pom were 13.9 and 11.2, respectively, with a broad range of concentrations showing a productive interaction (Figure 3B, D). Notably, in an IMiD-resistant relapsed patient sample, the combination of omacetaxine and Pom was even more synergistic and re-sensitized the MM cells to the IMiD (22.7 δ-score, Figure 3E-F). In contrast, omacetaxine combined with the PI bortezomib showed a lack of synergy when screened in the same manner (Supplementary Figure 5A-B). Omacetaxine combined with dexamethasone was synergistic in only one out of three MM cell lines tested (Supplementary Figure 5C-D). Thus, when combined with omacetaxine, IMiDs displayed unique and consistent synergy that was not observed with other anti-myeloma drugs tested.

Figure 3. Combination treatments with omacetaxine and immunomodulatory drugs.

(A) Myeloma cell line viability after 96 h treatment with 20 μM lenalidomide (Len) and 15 nM omacetaxine (Oma) as single agents or in combination. Cell viability is graphed as % normalized (Norm) to untreated controls. (B) ZIP synergy plot of lenalidomide and omacetaxine combinations matrix in MM.1S cells after 96 h treatment. (C) Myeloma cell line viability after 96 h treatment with 20 μM pomalidomide (Pom) and 15 nM omacetaxine as single agents or in combination. (D) ZIP synergy plot of pomalidomide and omacetaxine combinations matrix in MM.1S cells after 96 h treatment. (E) Combination treatment with pomalidomide and omacetaxine was synergistic and restored pomalidomide sensitivity in an MM patient sample. (F) ZIP synergy plot of pomalidomide and omacetaxine combinations matrix in patient sample HTB-576, δ-score = 22.7.

To extend our findings to an in vivo model, we evaluated omacetaxine using a firefly luciferase-expressing myeloma cell line, MM.1S (luc-MM.1S), to generate xenografts in NSG mice. MM.1S was chosen for its similarity to primary MM compared to other available cell lines.³⁶ First, we sought to study the pharmacodynamics of omacetaxine in this model. Mice were injected with luc-MM.1S cells and allowed to establish disease, followed by treatment with 1, 2, or 3 mg/kg omacetaxine by intraperitoneal (IP) injection, followed by puromycin injection one hour later, and sacrifice 1.5 hours later, with bone marrow harvest and OP-puro incorporation by flow cytometry (Figure 4A). In this study, we identified a dose-dependent decrease in the protein translation rate in the luc-MM.1S cells, with the greatest effect occurring at 3 mg/kg (Figure 4B). These results were used to design a study to determine the benefit of combining omacetaxine with pomalidomide treatment in vivo.

Figure 4. In vivo mouse modeling of multiple myeloma with omacetaxine treatment.

(A) Schematic showing the myeloma luciferase xenograft model. NSG mice were injected with 500,000 MM.1S cells, and the engraftment was confirmed after 30 days via IVIS bioluminescence imaging. Mice were injected with vehicle or with 1 mg/kg, 2 mg/kg, or 3 mg/kg omacetaxine (Oma). After 1 h, 500 μg puromycin (Puro) was given IP, and the proteins were labeled for 1.5 h before the bone marrow was harvested and stained with anti-puromycin Alexa Fluor 488 antibody. (B) Percentage normalized (Norm) to vehicle treated controls of the puromycin MFI of isolated MM.1S cells from the xenograft model with increasing doses of omacetaxine. (C) Schematic illustrating the design of the combination treatment survival study. NSG mice were injected with 500,000 MM.1S cells. Thirty days after cell injection, mice were injected with vehicle, 1 mg/kg omacetaxine, 8 mg/kg pomalidomide (Pom), or the combination (combo) of 1 mg/kg omacetaxine and 8 mg/kg pomalidomide. (D) Luciferase imaging of MM.1S xenografts, day 1 represents first day of drug treatment. (E) Kaplan-Meier survival of the in vivo MM model. (F) Comparison of treatment arms showing hazard ratios (HR) and p values between each group. Statistics were calculated from data in (E) using cox-proportional hazard model.

We next tested combination treatment with omacetaxine and pomalidomide using the in vivo model with treatment starting when disseminated disease was detectable. To evaluate the benefit of the drug combination, lower dose levels of omacetaxine (1 mg/kg) and Pom (8 mg/kg) treatment were used in the in vivo experiment. Four study arms were used to initiate the treatments consisting of vehicle control, omacetaxine monotherapy, pomalidomide monotherapy, and omacetaxine/pomalidomide combination therapy IP daily Monday-Friday (Figure 4C). Based on BLI monitoring, the disease development occurred more slowly in the omacetaxine/pomalidomide combination therapy arm (Figure 4D, Supplementary Figure 6A-B). Mouse survival was also most significantly extended in the combination therapy arm compared to the vehicle control arm (Figure 4E-F).

Omacetaxine and Pomalidomide Induce a Double Hit on IRF4 and c-MYC

As both omacetaxine and pomalidomide directly affect protein levels, with omacetaxine acting by blocking protein production and pomalidomide acting through selective protein degradation, we examined the effects of the combination treatment on the proteome. We started with whole cell proteomics of MM cells comparing the single agent treatments to the combination treatment at a timepoint before substantial apoptosis or cell death began. Although omacetaxine is rapidly cytotoxic to MM cells, the cytotoxicity of pomalidomide occurs later, with apoptosis and cell death beginning at 24 h (Supplementary Fig 7A). In addition, the glycolysis was reduced most drastically in MM cell lines after combination treatment (Supplementary Figure 7B-D). For optimal detection of protein changes by these drugs in proteomic analyses, MM.1S cells were treated in triplicate with omacetaxine for 2.5 h, with pomalidomide for 24 h, or with the staggered combination treatment and compared to vehicle controls. Proteomic analyses identified 2,015 proteins, of which 30 were significantly depleted (P < 0.05, fold change decrease > 2) by omacetaxine, 25 by pomalidomide and 53 by the combination (Supplementary Tables 2-4).

We next examined the proteins that were most differentially changed in the treated cells compared to the vehicle control cells. By unsupervised clustering and PCA analysis, the treated cells separated differentially into three groups that were distinct from the vehicle control group and from each other (Supplementary Figure 8A-B). The top 25 most differentially affected proteins are shown by heatmap in Figure 5A. IGL1 and JCHAIN, which are components of the IgA protein produced by the MM.1S cells, were among the proteins that were most differentially downregulated by omacetaxine alone (Figure 5B-C). IRF4 was among the proteins that were most differentially downregulated by the combination treatment (Figure 5D, Supplementary Figure 8C-E). IRF4 stood out because it is critical to MM cell survival and a known downstream target of IMiD-mediated Ikaros degradation.^28,37,38 Since we used a staggered treatment schedule for proteomics in which pomalidomide treatment was administered for 24 h and omacetaxine added during the last 2.5 h, we repeated the cytotoxicity measurement and found that the combinations remained synergistic (Supplementary Fig 8F). Overall, the stepwise decrease in IRF4 between the single agent and combination treatments supports the idea that the combination may act as a double hit on the levels of this protein.

Figure 5. Proteomic analysis of omacetaxine treatment alone and in combination with pomalidomide.

(A) Heat map of the z-scores for the top 25 most differentially affected proteins in MM.1S cells following omacetaxine (Oma, 2.5 h), pomalidomide (Pom, 24 h), and combination (Pom 24 h, Oma 2.5 h) treatments, utilizing the Ward clustering algorithm and Euclidean distance measure. (B-D) Proteomic spectral read value comparison of the IGL1, JCHAIN, and IRF4 levels across treatment groups. Data represent means ± SD, comparisons by ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

In MM, IRF4, and c-MYC form a positive feedback loop that propels malignant survival and proliferation.^10,37 Although c-MYC was not detected, it may have been below the resolution of semi-quantitative proteomics, which can miss short half-life proteins. However, FUBP1, a transcription factor known to upregulate c-MYC³⁹, was among the most significantly downregulated proteins by the combination treatment (Figure 5A, Supplementary Figure 8G). Thus, we envisioned a model in which omacetaxine and IMiDs synergize to cause an even greater reduction in IRF4 and c-MYC levels (Figure 6A). To test this model, we incubated MM.1S cells with omacetaxine and pomalidomide alone and in staggered combination and observed a greater loss of c-MYC and IRF4 compared to single agents as measured by immunoblot and intracellular flow cytometry (Figure 6B-C). To further test the model, we evaluated whether omacetaxine and pomalidomide would be synergistic with JQ1, a BET inhibitor which downregulates c-MYC transcription.⁴⁰ JQ1 was synergistic with both omacetaxine and pomalidomide in MM cell lines (17.56 and 21.18 δ-scores, respectively, Figure 6D-E). In summary, omacetaxine and IMiDs are synergistic in MM cells and cooperate to elicit a more substantial downregulation of the IRF4/c-MYC pathway than either drug alone, creating an attractive and clinically testable regimen for patients with relapsed/refractory MM.

Figure 6. Omacetaxine is synergistic with anti-MYC targeted therapy.

(A) Model of omacetaxine and IMiD combination therapy to cooperatively downregulate the IRF4/c-MYC axis in MM cells. Green arrows indicate IMiD effect, red arrows indicate omacetaxine effect, and purple arrows indicate combination treatment effect. (B) Immunoblots of MM.1S cells after treatment with omacetaxine (50 nM) and pomalidomide (10 μM) alone or in combination from 0-24 h. (C) Relative IRF4 and c-MYC expression measured by intracellular flow cytometry in MM.1S cells after 48 h pomalidomide (Pom, 10 μM), 4 h omacetaxine (Oma, 50 nM), or the combination. (D) In vitro combination treatment of the BET inhibitor JQ1 (50 nM), which also downregulates c-MYC, with omacetaxine (40 nM) in the MM.1S cell line for 96 h including ZIP synergy analysis. (E) In vitro combination treatment of JQ1(100 nM) with pomalidomide (1.25 μM) in the cell line U266 for 96 h including ZIP synergy analysis. Data represent means ± SD, comparisons by ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

DISCUSSION

Despite improvements in therapies over the past two decades, MM remains an incurable blood cancer. Nearly all MM patients experience relapse and eventual resistance to current therapies. The relapsed and refractory multiple myeloma (RRMM) setting continues to require new therapeutic options, particularly therapies with unique mechanisms of action that are distinct from those of existing therapies. One way this could be accomplished is through development of new small molecule classes that exploit cellular processes (e.g. protein synthesis) and/or molecular pathways (e.g. IRF4 and c-MYC) that are uniquely upregulated in malignant plasma cells.

In oncology, omacetaxine has a unique mechanism of action in which it binds to the A-site cleft of ribosomes and inhibits protein biosynthesis. In the context of multiple myeloma, several independent groups have now shown that the inhibition of protein translation has selective and potent cytotoxic effects.^19-21,23,25 This approach may be tumoricidal through diminution of short-lived oncoproteins such as MCL1 and c-MYC.^23-25,41 Here, we extend the previous preclinical studies showing omacetaxine activity in MM cell lines by documenting broad efficacy in primary samples across the disease spectrum and describing clinically applicable drug combination with IMiDs. MM is a particularly attractive disease for development of this strategy in light of the significant unmet medical need in the growing patient population that is refractory to currently available drugs. Together with the previous reports, our study further supports clinical testing of this approach.

We first became interested in omacetaxine as a potential means to exploit the MCL1-dependence that has emerged as underlying MM pathogenesis. We have developed a high-throughput, flow cytometry-based approach termed myeloma-drug sensitivity testing (My-DST) to test primary samples.³⁰ With My-DST, omacetaxine treatment showed specific, dose-dependent cytotoxic effects on patient MM cells, with no appreciable effect on the normal bone marrow cells. In a substantial sized cohort that included 51 MM patient samples, treatment with 50 nM omacetaxine showed anti-myeloma activity in the majority of samples and was independent of resistance to IMiDs or PIs. This ex vivo dose of 50 nM (27.3 ng/mL) is within the achievable human plasma levels of 36.2 ng/mL.⁴² Surprisingly, we found that both MCL1-dependent and -independent samples were susceptible to omacetaxine. To help understand this broad activity, we examined protein translation and found almost ubiquitously higher levels of protein translation in MM cells compared to background MNCs. Based on these results using primary samples, our findings show that omacetaxine retains its efficacy in PI/IMiD refractory MM, a finding that cannot be gleaned from cell line data.

In the current clinical approach to MM, combination therapies are utilized almost exclusively rather than single agents.⁴³ The benefit of this combinatorial approach is likely still due to the independent actions of the drugs, rather than mechanistic synergy in most cases.⁴⁴ Although true mechanistic drug synergism may be rare in MM, it may be very valuable in combating drug resistance. In our evaluation of combination treatments using clinically available agents together with omacetaxine, the IMiD-based combinations stood out as synergetic. Based on our proteomics and targeted protein measurements, we found that this synergy made mechanistic sense, since the crucial MM survival factors IRF4 and c-MYC were most substantially downregulated after the combination treatment. Targeting c-MYC in MM via translation inhibition is also independently supported by the previous study by Manier and colleagues.²³

Although the importance of c-MYC and MCL1 for MM survival is well supported, approaches that target these oncoproteins have yet to demonstrate clinical activity. Targeted MCL1 inhibitors have recently emerged and are being tested in phase I clinical trials as single agents (NCT03218683), but one is currently on FDA placed hold for possible cardiac toxicity (NCT03465540).^35,45 Pharmacologic targeting of c-MYC has been challenging, but recently BET inhibition was supported as one possible approach.⁴⁰ A phase I study of BET inhibition in MM patients is also underway (NCT03068351). Whether MCL1 and BET inhibitors will be safe and effective is not yet clear, whereas omacetaxine is FDA approved and tolerable in humans. In this study, we found that MCL1 inhibition had less anti-myeloma activity than omacetaxine treatment. For protein translation inhibition, other studies have been published supporting its potential as a new avenue for MM treatment,^23-25 but no clinical trials have yet opened using this approach. Thus, omacetaxine may have the advantages of being multi-targeted and readily translated into patient testing.

Based on the broad preclinical activity of omacetaxine in MM as well as its strong and consistent synergy with immunomodulatory drugs, a dedicated clinical trial of this approach in patients with MM should be conducted. In the clinical trials, the measurement of the protein translation rate in MM cells could serve as a biomarker for those patients most likely to respond. Based on the prior clinical experience in patients with CML, omacetaxine has favorable bioavailability in humans with a suitable side effect profile.⁴⁶ Primary adverse events observed included hematologic events, chiefly thrombocytopenia, grade 1-2 infections, and gastrointestinal side effects.⁴⁷ In addition, two case reports of MM patients treated with omacetaxine-based combination reported responses.⁴⁸ Thus, omacetaxine is known to be bioavailable and safe in patients, suggesting that administration will be straight forward in MM and may be tolerable in drug combinations as well. To achieve this, we have designed a phase I clinical trial to evaluate omacetaxine as a single agent and in combination with pomalidomide for patients with relapsed/refractory multiple myeloma.

TRANSLATIONAL RELEVANCE.

Even with an abundance of treatment options available to multiple myeloma (MM) patients, including drugs ranging from proteasome inhibitors to monoclonal antibody therapy, patients still inevitably develop drug resistance. This relapsed and refractory MM setting continues to require new therapeutic options, the most promising of which have unique mechanisms of action. Protein translation inhibition represents a new class of drugs for MM patients. Here we show that by inhibiting protein synthesis with the translation inhibitor omacetaxine, specific anti-myeloma activity was observed in ex vivo MM cells from patient samples (n=51), including samples that were resistant to currently available drugs. This was further confirmed using an in vivo mouse model of MM. Considering the preclinical evidence we describe, and the fact that omacetaxine is already FDA approved for the treatment of chronic myeloid leukemia, we believe that repurposing this agent should be pursued for patients with multiple myeloma.