Proteasomal Inhibition by Ixazomib Induces CHK1 and MYC Dependent Cell Death in T-Cell and Hodgkin Lymphoma

Abstract

Proteasome regulated nuclear-factor kappa-B has been shown to be important for cell survival in T-cell lymphoma (TCL) and Hodgkin lymphoma (HL) models. Several new small molecule proteasome inhibitors are under various stages of active preclinical and clinical development. We completed a comprehensive preclinical examination of the efficacy and associated biological effects of a second-generation proteasome inhibitor, ixazomib, in TCL and HL cells and in vivo SCID mouse models. Our results demonstrated that ixazomib induced potent cell death in all cell lines at clinically achievable concentrations. Additionally, it significantly inhibited tumor growth and improved survival in TCL and HL human lymphoma xenograft models. Through global transcriptome analyses, proteasomal inhibition showed conserved overlap in down-regulation of cell cycle, chromatin modification, and DNA repair processes in ixazomib sensitive lymphoma cells. The predicted activity for tumor suppressors and oncogenes, the impact on “hallmarks of cancer”, and the analysis of key significant genes from global transcriptome analysis for ixazomib strongly favored tumor inhibition via down-regulation of MYC and CHK1 its target genes. Furthermore, in ixazomib-treated lymphoma cells, we identified that Chk1 was involved in the regulation of MYC expression through chromatin modification involving histone H3 acetylation via chromatin immunoprecipitation (ChIP). Finally, using pharmacologic and RNA silencing of CHK1 or the dependent MYC related mechanism, we demonstrated synergistic cell death in combination with ixazomib treatment. Altogether, ixazomib significantly downregulates MYC and induces potent cell death in TCL and HL and we identified that combinatorial therapy with anti-Chk1 treatment represents a rational and novel therapeutic approach.

INTRODUCTION

Understanding the pertinent and functional biologic oncogenic pathways of inhibition and mechanisms of resistance are critical in order to optimally integrate novel therapeutics into cancer treatment paradigms. Increased proteasomal activity is a frequently observed phenomenon in malignant cells; hence, proteasome inhibition may potentially block cell proliferation and result in the inhibition of tumor progression (1). Bortezomib (Velcade®) was the first in class proteasomal targeted drug that is currently FDA approved for the treatment of newly-diagnosed and relapsed/refractory multiple myeloma and mantle cell lymphoma (2). Next-generation proteasome inhibitors, such as ixazomib, are currently under pre-clinical and clinical investigations. These novel agents were developed with an objective of enhancing efficacy and improving the tolerability of anti-proteasomal therapeutics (3–5). Ixazomib is an investigational proteasome inhibitor currently in clinical trials in hematological malignancies and early reports indicate promising clinical activities in follicular and T cell lymphoma (TCL) (6), however its biological activity in TCL and Hodgkin lymphoma (HL) are largely unknown and the biologic mechanisms are not well defined.

TCLs represent a heterogeneous group of aggressive non-Hodgkin lymphomas with overall poor prognosis (7). Further discovery of its tumor biology is needed and additional novel targeted therapeutic agents are desired. HL is generally a curable malignancy by conventional chemotherapy, however there continues to be a subset of patients with refractory disease or relapse that succumbs to this disease. Additionally, there remains an unmet need to identify targeted, less toxic therapy for the treatment of HL in order to decrease the continued acute and long-term toxicities associated with cytotoxic chemotherapy and radiation.(7–9) Moreover, multiple pre-clinical studies strongly indicated that proteasome dependent nuclear factor kappa-B (NFκB) may be a “master switch” of Hodgkin Reed Sternberg (HRS) cells and it has been shown to be critical for cell survival in HRS and also TCL models (10–15). While proteasome is a rationale therapeutic target for the treatment of TCL and HL, clinical studies in TCL and HL patients treated with bortezomib were associated with modest clinical benefit (16, 17). With the development of ixazomib as second generation proteasomal inhibitor with superior pharmacodynamic, pharmacokinetic and tumor inhibitory properties in lymphoma compared with bortezomib,(18, 19) interest in the treatment of TCL and HL is renewed.

We utilized in vitro and in vivo tumor models to understand the biologic mechanisms of action and antitumor activity of ixazomib in TCL and HL, at clinically relevant concentrations. Through global transcriptome and network analysis, we describe the impact of ixazomib on biological pathways and tumor progression in TCL and HL cells. Furthermore, our investigation outlines mechanisms of CHK1-dependent MYC-regulated cell death occurring via ixazomib and the associated critical implications impacting cell signaling and drug sensitivity. The impact of ixazomib on CHK1 and MYC is significant in the context of other previous studies indicating an existence of co-operative relationship between MYC and CHK1 as an important factor in driving lymphomagenesis (20). Further sensitivity to CHK1 inhibition has also been reported in lymphoma cells with MYC overexpression (21). Thus, with the ability to impact MYC and CHK1 as key driving mechanisms of tumorigenesis in lymphoma, ixazomib is a potential appealing anti-lymphoma agent.

METHODS

Cell culture, reagents and transfections

HL cell lines L540 and L428 and, TCL cell lines HH, Hut78 and Jurkat were grown in RPMI 1640 consisting of 10% heat inactivated fetal bovine serum and penicillin/streptomycin (Mediatech, Manassas, VA) under 5% CO₂ and 37°C. Cell lines were authenticated using short tandem repeat (STR) profiling service provided, by ATCC (Manassas, VA). Ixazomib was kindly provided by Takeda Pharmaceuticals, Inc. Belinostat, JQ1, and the CHK1 inhibitor, AZD7762, were purchased from Selleck Chemicals (Houston, TX). Non-targeting or MYC siRNA were obtained from GE Healthcare, (Lafayette, CO) and transfection was performed using Nucleofector device and reagent kit L (Lonza, Walkersville, MD).

MTT and apoptosis

For MTT assay and Annexin V apoptosis detection, performed as described before (22). IC₅₀ values and combination indices for drug treatment were derived using Calcusyn Version 2.1 Software (Biosoft, Ferguson, MO).

Lymphoma xenografts

For examination of the in vivo effect of ixazomib, human lymphoma xenografts derived from Jurkat (TCL) or L540 (HL) grown in SCID mouse models as described before,(22) were treated with either saline (control), or ixazomib (0.36 or 0.72mg/kg) by intraperitoneal (i.p.) injections daily for 5 days for three weeks consisting eight mice per group. Animals observation, tumor and body weight measurements, and statistical analysis using GraphPad Prism 5.0, (GraphPad Software, Inc. La Jolla, CA), were performed as described before (22). To quantify tumor growth, we used nonlinear regression to fit the tumor growth phase data with curves of the form V= be^ct, where V is tumor volume and t is time (23). Here, c is interpreted as the tumor growth rate once the tumor starts to grow more or less exponentially; b is interpreted as the extrapolated “effective” volume at the implant time, t =0.

Western blot

Preparation of protein lysates and Western blot was performed, as previously described (22). Primary antibodies against Myc, total or phospho Chk1 (Ser 345), total or acetylated Histone H3, β-actin, HDAC3, p62, Cathepsin D total and cleaved caspase-3, 8, 9, PARP and β-actin were purchased from Cell Signaling Technology (Beverly, MA).

Transcriptome analyses

Jurkat, L540, and L428 cells were treated in triplicates with 25nM ixazomib for 24 hours, RNA was isolated using RNeasy Minikit (Qiagen) and microarray experiment was performed using Affymetrix Human Gene Chip 2.0 (Jurkat and L540), or Human HT 12 Genechip Illumina (L428). The raw expression data from these experiments are available at NCBI Gene Expression Omnibus (GEO) database, with following identifiers GSE66417 (Jurkat/L540) and GSE66415 (L428). Data for Jurkat and L540 cell lines were background adjusted and quantile normalized using RMAExpress (24). Statistically relevant genes were determined by applying LIMMA (25) with a FDR < 0.05. Data for the L428 cell line (part of a larger dataset which was not used in this manuscript) was corrected through normalization of the housekeeping genes, quantile normalized, and then statistically relevant genes were determined with one-way ANOVA analysis with FDR <0.05. Details on the pathway analysis, networks, and the unbiased method to determine the key significant genes are previously published in (23, 26, 27).

ChIP and PCR assay

Sample preparation was performed using Pierce Agarose ChIP kit, Thermo Scientific, (Rockford, IL), quantitative real-time PCR was performed using primer set (#PPH00100B) SABiosciences, Valencia, CA, designed to amplify human MYC promoter region proximal to transcription start site with SYBR green mastermix (ROCHE, Indianapolis, IN), and using Roche LightCycler I. For chromatin IP, Chip grade antibodies, Acetyl Histone H3 K9 (#17-658) purchased from EMD Millipore, (Bellerica, MA), anti-RNA polymerase II and normal rabbit IgG supplied in Pierce Agarose ChIP kit used as positive and negative control, respectively.

RESULTS

In vitro and in vivo efficacy with anti-proteasomal therapy

Cell viability following exposure to ixazomib (12.5–1000nM) for 72 hours in TCL cell lines (Jurkat, Hut78, HH) and HL cell lines (L428, L540) resulted in a dose-dependent decrease in cell viability in all cell lines (Figure 1A–B). L540 cells were observed to be most sensitive with associated 50% inhibitory effective dose (ED) concentration ED₅₀ of 25nM compared with HH (41nM), Hut78 (52nM), Jurkat (38nM), and L428 (117nM). Treatment with ixazomib resulted in accumulation of ubiquitinylated protein as would be expected with proteosomal inhibition (Figure 1C). Additionally, there were induction of apoptosis in all lymphoma cell lines as detected by western blot analysis of caspase 3, 7, 8, and 9, and PARP cleavage, except in L428 (Figure 1C); this was confirmed with ixazomib-treated lymphoma cells using Annexin/PI staining and flow cytometry (Figure 1D).

Figure 1. Proteasomal inhibition induces potent in vitro cell death and inhibits in vivo tumor growth in human lymphoma xenografts.

A–B. Dose dependent increase in cytotoxicity with ixazomib in TCL and HL lymphoma cell lines by MTT assay, at 72 hours. C. Western blot analysis of ixazomib treated TCL or HL cell lines show accumulation of ubiquitinylated protein, activation of apoptotic markers, at 24 hours. D. Bar graph shows dose dependent significant increase in Annexin-V positive apoptotic cells in ixazomib treated lymphoma cells (*), P<0.05 or (**) P<0.0001). Tumor bearing SCID mice treated with ixazomib (0.72 mg/kg) showed significant reduction in the tumor volume (represented as line graph, P<0.001, Two way repeated ANOVA), and Kaplan Meier analysis show significant increase in survival compared to controls (P<0.001) in the experiments performed with (E–F) Jurkat or (G–H) L540 derived tumor xenografts. (I) Tumor growth rate plot showing the effect of ixazomib treatment on tumor progression over the entire duration of assessment, with significant decrease in growth rate (P<0.001) in L540, while in Jurkat lag in tumor growth is noted.

The in vivo efficacy of ixazomib was investigated using tumor xenografts derived using either Jurkat or L540 cell lines. Results from Jurkat-derived xenograft experiments showed that after four weeks of ixazomib administration, there was a significant reduction in the average approximate size of the tumor (1094 ± 76mm³) with 0.36 mg/kg or (582 ± 45mm³) with 0.72 mg/kg compared to the average size of tumor (1465± 224mm³) in the vehicle treated control group (p<0.0001) (Figure 1E). Kaplan Meier survival analysis showed a significant improved survival in SCID mice consisting Jurkat xenograft treated with ixazomib compared to control group (p<0.0001) (Figure 1F). Treatment with ixazomib in L540 xenograft tumor bearing SCID mice also resulted in a significant reduction in the average size of the tumor (821 ± 54 mm³) with 0.36mg/kg or (290 ± 5 mm³) with 0.72mg/kg compared to vehicle administered control group (1128 ± 162 mm³), at the end of four weeks (p=<0.001) (Figure 1G). Kaplan Meier survival analysis showed a significant increase in survival with ixazomib treatment compared to control groups (p<0.0001) (Figure 1H).

We also analyzed detailed tumor growth kinetics by comparing tumor volumes measured prior to, during, and at the completion of treatment. Ixazomib resulted in decreased average tumor growth rate, 0.045 ± 0.0060 with 0.36mg/kg and significantly (P=0.002), 0.017 ± 0.0060 with 0.72mg/kg), compared with the growth rate 0.049 ± 0.0045 in the vehicle administered control group (Figure 1I) consistent with inhibition of tumor progression in L540. This indicates there will be steady decrease in tumor growth with increasing doses of ixazomib. The kinetics of tumor growth in the Jurkat xenograft, however, showed no significant differences in growth rate (0.076 ± 0.012 with 0.36mg/kg and 0.056 ± 0.015 with 0.72mg.kg) compared with the growth rate of 0.052 ± 0.006 vehicle administered control group, suggesting that ixazomib is causing a lag time delay in the tumor growth rather than any significant change in the growth rate. It should be noted that this prediction is obscured by large variances in the growth rates of tumor observed in the ixazomib-treated cohorts (Figure 1I). Although ixazomib treatment appeared to affect the kinetics of the tumor growth by causing a lag time delay in the Jurkat-derived xenografts, comparison of average tumor size resulted in overall reduction in the tumor burden and improved survival benefit (Figure1E and 1H) indicating anti-tumor activity of ixazomib in this model. The results from in vitro and in vivo experiments demonstrate that proteasomal inhibition via ixazomib is associated with strong activity against TCL and HL.

Transcriptome analyses of the impact of proteasomal inhibition on biological pathways and “hallmarks of cancer”

Global transcriptome analyses following ixazomib exposure showed significant gene changes with a 1.2 fold-change for Jurkat (508 genes), L540 (4765 genes), and L428 (423 genes) (Figure 2A) with a FDR <0.05. L540 with the highest significantly differential expressed genes was the most ixazomib sensitive HL line (Figure 1A) with significant inhibition of tumor growth (Figure 1I). Gene expression with ixazomib compared with untreated control showed a total of 40 overlapping genes with a 1.2 fold-change difference in Jurkat, L540, and L428 cells, with 326 overlapping genes in Jurkat and L540, and 212 overlapping gene sets in L428 and L540 HL cells (Figure 2B). The overlap based on common significantly regulated genes in Jurkat, L540, and L428 cells treated with ixazomib showed conserved up-regulation of ubiquitin proteasome components PSMB3, PSMB6, PSMC4, PSMD6, UBE2H, UBFD1 and lysosome associated factors ATG4A, CD68, PSAP and SQSTM1 (also known as p62) (9), (Figure 2C) and the gene expression changes were confirmed by quantitative real-time PCR based assay (Supplemental Figure S1).

Figure 2. Gene expression profiling identifies critical genes, signaling pathways, and regulators affected by proteasomal therapy.

A. Average signal log₂ fold-change comparing Ixazomib 24 hours versus control for 25nM ixazomib treatment in Jurkat, L540 and L428 cells. Whiskers show the range of the outliers, with max and min values as O and the 1 and 99^th percentile outliers as X. Individual data points are shown on the left of box plots as filled circles. Dotted red lines show the 1.2 fold-change cutoff. B. Venn diagrams of the genes with 1.2 up- and down-regulated fold changes for comparisons between ixazomib 24 hours vs. Control for Jurkat, L540 and L428 cells. C. Scatter plot of average signal log₂ fold-change comparing common significantly regulated genes between ixazomib 24 hours vs. Control for 25nM ixazomib treatment in Jurkat, L540 and L428 cells. Dotted red lines show the 1.2 fold-change cutoff.

Upstream regulator analysis performed using Ingenuity Pathway Analysis (IPA) and in conjunction with literature evidences reporting impact on tumor progression, (Figure 3, Table S1) summarized the effects of ixazomib on tumor dynamics. Ixazomib treatment in Jurkat and L540 appeared to have shifted the balance of upstream regulators associated with tumor promotion and tumor suppression in favor of tumor inhibition (Figure 3A), supporting the tumor inhibitory effects of ixazomib observed with tumor xenografts (Figure 1I). Conversely, the responses in L428 with ixazomib appeared to favor tumor progression (Figure 3). Next, the status of upstream regulators was mapped on their functional contextual relationship to classical “hallmarks of cancer” (28, 29). Results from this evaluation show that ixazomib overwhelmingly impacts 9/12 “hallmarks of cancer,” (Figure 4A), providing clues to tumor inhibitory functions of ixazomib in L540 (Figure 3A). In contrast, ixazomib treatment in L428 appears to promote the ‘self-sufficiency in growth signal’ favoring tumor progression (Figure 4B). Although additional experiments for a functional validation of these predictions are required, it must be noted that there was an apparent lack of apoptotic activity in L428 cells despite proteasomal inhibition with ixazomib (as detected as accumulation of ubiquitinylated protein) (Figure 1C). In fact, these cells demonstrated the lowest sensitivity to ixazomib compared with other cell lines tested (Figure 1A & B). Interestingly, MYC inhibition represented among several “hallmarks of cancer” present in L540 is notably absent in L428 with ixazomib treatment (Figure 4) and further validated by quantitative real-time PCR based assay (Supplemental Figure S1). MYC is functionally associated with several oncogenic mechanisms associated with tumor progression,(30) and inhibition of MYC with ixazomib treatment could be an important event in the antitumor activity of ixazomib.

Figure 3. Common upstream regulators determined by Ingenuity Pathway Analysis (IPA) software from the significant genes with anti-proteasomal treatment.

A. A schematic of the activation states of the upstream regulators illustrating the balance between the tumor promoters (text in yellow) and tumor suppressors (text in white and underlined) with a predicted activation (circled in orange) or predicted inhibition (circled in blue). B. Scatter plot of the common significant upstream regulators (with activation Z-scores > 2 or < −2) between ixazomib 24 hours treatment versus controls for Jurkat, L540, and L428 cells. Non-significant genes for L428 are also shown as grey dots. Dotted red lines show the cutoff for significantly regulated upstream regulators.

Figure 4. Effects of anti-proteasomal treatment on the “hallmarks of cancer.”.

A–B. A schematic of the “hallmarks of cancer” overlaid with activation states of the upstream regulators illustrating the balance between the tumor promoters (text in red or blue) and tumor suppressors (text in red or blue, and underlined) with a predicted activation (text in red) or predicted inhibition (text in blue), showing the effect on “hallmarks of cancer” inhibition (shaded in dark/light green) or activation (shaded in red/orange) in the ixazomib treatment versus controls for L540, and L428 cells.

Gene Set Enrichment Analysis (GSEA) revealed conserved upregulation of biological pathways representing protein localization, proteasome, vesicle transport, apoptosis, oxidative stress, NFκB and catabolic processes with ixazomib in both L540 and L428 cells (Figure 5A, Figure S2, Table S2 and S3). This included conservation of biologic pathways with gene expression analysis using different and higher doses of ixazomib in L428 and L540 (data not shown). While biological pathways associated with DNA repair, mitotic cell cycle and microtubule organization oppositely regulated between L540 and L428 HL cells, such patterns of biological responses to ixazomib were consistently observed with GSEA performed using multiple reference databases (KEGG, Reactome and Biocarta) (Figure S2, Table S2 and S3). Additionally, GSEA predicting overall downregulation of MYC pathway with ixazomib in L540 or Jurkat (Figure S1C) as validated by quantitative real-time PCR based assay (Supplemental Figure S1).

Figure 5. GSEA of transcriptomic network and the significant key genes in responses to anti-proteasomal treatment.

A. Network representation of Gene Set Enrichment Analysis (GSEA) for GO C5 gene sets for Ixazomib 24 hours versus control for Jurkat, L540 and L428 cells, with L540 overlaid with either Jurkat or L428. Leading edge analysis with a FDR<0.05 determined significant gene sets enriched for each group. The size of each node reflects the amount of molecules involved for each gene set. The edge thickness (green lines for L540 and blue lines for either Jurkat or L428) represents the number of genes associated with the overlap of two gene sets (or nodes) that the edge connects. Clusters in each grouping were named according to common function. Upregulated gene sets denoted with red color and downregulated gene sets were denoted by blue color. B. Common significant key genes with the lymphoma cells treated with ixazomib. The interactions of the common key significant genes with symbols representing the biological function and colors denoting up or down regulation in gene expression, are described in the inserted legend. C. Schematic of the activation states of the common significant key genes illustrating the balance between the tumor promoters (text in blue) and tumor suppressors (text in black and underlined) with the status of up (circled in red) or down regulation (circled in green) in the gene expression experiment with ixazomib.

Sixteen common key significant genes were determined for Jurkat and L540 cells treated with ixazomib, and these were non-overlapping with L428 (Table S4). These key genes are considered as “core responsive genes” and were determined by comparing common genes involved in the predicted upstream regulator analysis and biofunction analysis through IPA with the genes involved in the GSEA analysis (Figure 5B). Surprisingly, the majority of these genes also have existing known interactions (Figure 5B). Analysis of these 16 key genes (shown in Figure 5B) using DAVID functional annotation and classification tool (31) identified 22 functional clusters that included mitotic cell cycle, apoptosis, proteolysis, metabolism, and lysosomal activity as key biological responses affected by ixazomib in Jurkat and L540 (Table S5).

Downregulation of MYC via proteosomal inhibition

Analysis of upstream regulators and GSEA with ixazomib transcriptome predicted MYC inhibition in L540 and Jurkat cells, but notably absent in L428 (Figure 3& 4) and validated by quantitative real-time PCR based assay (Supplemental Figure S1). MYC is an important regulator of cell cycle, DNA repair and replication, chromatin modification, inflammatory responses, lysosomal autophagy, metabolism and oxidative stress (32, 33) and these pathways are also enriched with ixazomib treatment (Figure S2). Therefore, we investigated to determine the biological responses of these enriched pathways using ixazomib treated panel of lymphoma cells. Results from our experiments showed that Myc protein levels decreased with increasing concentrations of ixazomib in all lymphoma cells, except in the relatively less sensitive L428 cell line (Figure 6A) consistent with transcriptional downregulation of MYC expression, as determined by quantitative real-time PCR based assay (Figure S1). A dose dependent increase in lysosomal activity-associated Cathepsin D cleavage were observed in most of the cell lines, with L428 showing increase in lysosomal autophagy related p62. Lysosomal activation could be relevant Myc downregulation with ixazomib because, previous study has shown Myc is a substrate in Cathepsin D dependent proteolysis (34), presumably this activation of lysosomal proteolysis by ixazomib could be relevant to downregulation of Myc protein levels.

Figure 6. Myc downregulation by ixazomib.

A. Western blot analysis show the dose dependent effects of ixazomib on Myc protein and the events associated with lysosomal activity, cell cycle and chromatin modification responses. B. Electron microscopic findings with ixazomib treatment show presence of vacuolar structures with light to dense amorphous granulations, representing lysosomal bodies (arrows), localized predominantly in the cytoplasm and occasionally present in the perinuclear or nuclear region. Cytoplasm is represented by “C” and nucleus is represented by “N” in the images. C. Inhibition of lysosomal proteases with E64/Pepstatin, potentiates downregulation of Myc and acetylation of Histone H3 and increases caspsase-3 and PARP cleavage, detected by western blot. D. Box plot based on average signal log₂ fold-change comparing ixazomib 24 hours versus control for 25nM ixazomib treatment in Jurkat, L540 and L428 cells. Dotted red lines show the 1.2 fold-change cutoff for the differentially expressed MYC and CHK1 related genes. E. Bar Graph show representation in percentages of MYC or CHK1 regulated differentially expressed genes within the entire set of MYC or CHK1 regulated genes, among the entire transcriptome and overlap between these differentially expressed gene sets in ixazomib treated Jurkat, L540 and L428 cells.

Proteasomal inhibition has been previously shown to inhibit DNA damage response to IR via inhibition of Chk1 phosphorylation (35) and our results from GSEA show downregulation of genes associated with DNA damage, DNA repair and cell cycle progression with ixazomib (Figures 5, S2, Tables S2 and S3). We examined inhibition of Chk1 phosphorylation as a marker for these responses (36) and observed inhibition of Chk1 phosphorylation in all cell lines with ixazomib treatment except in L428 (Figure 6A). Histone H3 acetylation, a marker of chromatin modification, is regulated by CHK1(37) and affect MYC expression;(38) was also decreased with increasing concentrations of ixazomib in Jurkat, HH and Hut78 and L540 cells, without significant effect in L428 cells (Figure 6A). Collectively, these results suggests that biological responses of MYC, lysosome, cell cycle and chromatin modification correlate with each other and are conserved in ixazomib TCL and HL cell lines, while such conservation was lacking in L428 cells, exactly as predicted from GSEA (Figure 5) and DAVID analysis (Table S5)

Lysosomal activity represents as alternate means for protein degradation and therefore, excess protein accumulation resulting from proteasomal inhibition is expected to trigger lysosomal function (39). Since MYC is also a substrate for lysosomal proteolysis,(34) we subsequently investigated the role of lysosomal activity and its relation to MYC. Analysis via electron microscopy revealed characteristic features representing lysosomal maturation in ixazomib treated cells (Figure 6B), however subsequent western blot analysis did not show evidence of autophagy determined based on mTOR and Beclin phosphorylation, and LC3A cleavage (data not shown). Further inhibition of lysosomal protease with E64d/Pepstatin did not result in Myc accumulation but rather led to accelerated reduction in Myc protein and Histone H3 acetylation, and enhanced apoptosis, detected as cleaved caspase 3 and PARP in ixazomib treated Jurkat and L428 cells (Figure 6C). There was no corresponding HDAC accumulation with decreased Histone H3 acetylation with lysosomal inhibition (Figure 6C) suggesting HDAC independent reduction of Histone H3 acetylation with ixazomib. Taken together, inhibition of proteasomal and lysosomal proteolysis, instead of resulting in Myc accumulation resulted in further loss of Myc protein, indicating a possible role for transcriptional regulation of MYC expression with ixazomib treatment.

Next, we focused our investigation on the cell cycle regulatory CHK1 in the context of MYC function, since MYC and CHK1 interactions is a known oncogenic mechanism in lymphomagenesis,(20) the role of CHK1 in transcriptional regulation via Histone H3 acetylation is known (37) and Histone H3 acetylation is required for MYC transcription (33). We examined genes regulated by MYC and CHK1 in ixazomib-treated cells and observed strong overlap in the target gene-sets regulated by MYC and CHK1. There was overall downregulation of MYC associated genes in both L540 (53%) and Jurkat (5%) and upregulation of MYC associated genes in L428 (6%) (Figure 6D&E). Similarly, there was overall downregulation of CHK1 associated genes in both L540 (47%) and Jurkat (11%) and upregulation of CHK1 associated genes in L428 (7%) (Figure 6D&E). By comparing MYC and CHK1 regulated genes, we noted the presence of overlapping gene-sets in L540 (20%), Jurkat (7%), and L428 (33%). Considering the strong overlap and the pattern of differentially expressed gene sets representing both MYC and CHK1, we narrowed our investigation to determine the biological relationship between these oncogenes.

MYC promoter binding and importance of CHK1

We investigated the effect of CHK1 inhibition on Histone H3 acetylation and MYC in L428 cells treated with ixazomib. We observed that CHK1 inhibitor (AZD7762) alone, or in combination with ixazomib, strongly downregulated Histone H3 acetylation, phosphorylation of CHK1 dependent Threonine 11,(37) on Histone H3, and MYC protein (Figure 7A). Furthermore, inhibition of histone acetyl transferase (HAT) by C646 also resulted in downregulation of MYC (Figure S3) indicating that CHK1 and Histone H3 acetylation are upstream in the regulation of Myc expression. Therefore, we performed chromatin immunoprecipitation (ChIP) using anti-acetylated Histone H3 and PCR assay for its binding on the MYC promoter region. We observed that ixazomib treatment resulted in decreased MYC amplification relative to untreated Jurkat control cells (Figure 7B) indicating reduced MYC promoter occupancy by acetylated Histone H3, as a possible mechanism for ixazomib dependent downregulation of MYC expression in the Jurkat cells. In contrast, we noted increased PCR amplification of MYC in L428 cells indicating higher promoter occupancy by acetyl Histone H3 suggesting an active transcription process with ixazomib (Figure 7B). Our conclusions based on the results from ChIP experiment showing decreased and increased promoter occupancy in Jurkat and L428 (Figure 7B), which are consistent with the patterns of MYC and MYC dependent gene expression observed in these cells (Figure 6A&D), indicate that MYC function is regulated at the level of transcription with ixazomib treatment. Since we observed that ixazomib treatment resulted in downregulation of Myc and CHK1 phosphorylation in all lymphoma cells, except in L428 cells, we chose to examine the consequences of blocking CHK1 on MYC expression in L428 cells. Previous study has demonstrated a direct role for CHK1 kinase activity in Histone H3 phosphorylation (Threonine 11) and stabilization of Histone H3 acetylation to facilitate transcription via retention of relaxed chromatin confirmation (37). Therefore, we performed ChIP PCR assay for MYC, in the presence of AZD7762 (CHK1 inhibitor) to determine whether CHK1 dependent histone modification affect MYC expression, and the results from our experiments show decreased MYC promoter occupancy by acetylated-Histone compared to untreated control; or in the presence of ixazomib in L428 cells (Figure 7C). Taken together these results demonstrated that CHK1 inhibition leading to decreased Histone H3 acetylation and MYC expression (Figure 7A) is associated with decreased promoter occupancy on MYC gene (Figure 7C), suggesting that CHK1 is actively involved in the transcriptional control of MYC in the ixazomib treated lymphoma cells.

Figure 7. CHK1 and Histone H3 acetylation-dependent Myc response to ixazomib.

A. Inhibition of CHK1 potentiates downregulation acetylation and phosphorylation of histone H3, and Myc protein with ixazomib. B. Chromatin IP with anti-acetylated Histone H3 and PCR for MYC promoter normalized with input DNA, show decreased promoter occupancy in Jurkat, and increased promoter occupancy in L428 cells with ixazomib treatment. C. Chromatin IP with anti-acetylated Histone H3 and PCR for MYC promoter normalized with input DNA, show that AZD7762 (i.e., CHK1 inhibitor) decreased acetylated Histone H3 promoter occupancy in L428 cells, with ixazomib treatment. D–F. Bar graphs show significant increase in Annexin V positivity (P<0.001) in ixazomib treated L428 cells in the presence of AZD7762 compared with all doses of ixazomib alone. Western blot analysis show treatment with JQ1 (i.e., bromodomain inhibitor) downregulates Myc and increases Caspsase-3 and PARP cleavage with ixazomib in Jurkat and L428 cells as well as via with MYC or CHK1 RNAi silencing results in increased PARP cleavage. G–J. Treatment of ixazomib in combination with AZD7762 or JQ1 for 72 hours, in L428 and the analysis of results from MTT assay show synergistic effect, (CI<1), represented as median does effect curve and combination indices graph, determined using Calcusyn software.

CHK1 or MYC inhibition synergizes with anti-proteasomal therapy

Considering that CHK1 phosphorylation and MYC expression were not affected by ixazomib treatment in L428, (Figure 6A & D), and L428 showed least sensitivity to ixazomib (by MTT and Apoptosis) (Figure 1B&C) compared to other lymphoma cells, we predicted that a combination CHK1 inhibitor and ixazomib is likely to downregulate Myc and restore ixazomib sensitivity in the L428 HL cells. Therefore, we treated L428 cells with increasing doses of ixazomib alone and in presence of CHK1 inhibitor, AZD7762, and observed a significant increase in Annexin-V positive apoptosis (P<0.001) in the combination of ixazomib with AZD7762 compared to controls (Figure 7D). Next considering that MYC downregulation is predicted as a prominent mechanism in ixazomib sensitivity, and we utilized JQ1 (bromodomain inhibitor, blocks MYC transcription) to inhibit MYC expression and observed potentiation of apoptosis, detected as cleaved PARP, both in Jurkat (TCL) and L428 HL cells in the presence of ixazomib (Figure 7E). Further these results were independently confirmed using RNAi mediated silencing of MYC or CHK1 (Figure 7F). Considering that MYC downregulation and co-targeting of MYC could be clinically relevant for ixazomib-based treatments, we finally investigated to determine whether such targeted drug combinations would be synergistic. Our results based on MTT assays, show that targeting CHK1 with AZD7762 (ED₅₀ 1.1µM) in the presence of ixazomib was synergistic (CI<1) in L428 HL cells (Figure 7G–H) and similarly, combination of JQ1 (ED₅₀ 5.5µM) with ixazomib also resulted in synergistic effect (CI<1) in L428 (Figure 7I–J), thus suggesting that AZD7762 or JQ1 dependent MYC downregulation will lead to synergistic cell death in ixazomib refractory L428 cells, with dominant MYC function.

DISCUSSION

The prominent substrates regulated by proteasome dependent turnover includes key proteins involved in the regulation of replication, transcription, translation, cell cycle, tumor suppression, signal transduction, apoptosis, metabolism among others (40). NFκB has been shown to be constitutively active in HL cell lines and also critical for HRS cell survival in xenograft studies (10, 11). Furthermore, NFκB has been shown to be highly overexpressed and responsible for tumorigenesis in both HRS and TCL models (14, 15). Despite these promising pre-clinical data, proteosomal inhibition with bortezomib had only modest clinical activity in HL,(16, 17) while more encouraging clinical activity has been reported in TCL (41, 42). To exploit the strong and functional presence of NFκB in HL and TCL, that which is regulated by the proteasome, we examined the effects of the novel 2^nd generation orally bioactive proteasome inhibitor, ixazomib, which has superior PK/PD compared with bortezomib (18). Considering also that early reports from ixazomib lymphoma clinical studies have been encouraging, (6) the results from our study provide the biological basis and the mechanisms of ixazomib activity.

Results herein demonstrated that ixazomib induced potent cell death and inhibited tumor growth in TCL and HL cells at clinically achievable nanomolar concentrations. Transcriptome analysis showed conserved and consistent biological responses with ixazomib in both TCL and HL cells. Further, the activation status of tumor suppressors and inhibition of oncogenes indicated strong potential for the inhibition of tumor progression with ixazomib in TCL and HL and substantial impact were predicted on overall global “hallmarks of cancer”. Although we observed conserved biological regulations with ixazomib treatment in Jurkat and L540 cells, we also noted that a lack of conservation with biological responses, associated with minimal impact on the “hallmarks of cancer” is related to poor sensitivity to ixazomib in L428 cells.

We postulated that examining the biological differences in response to ixazomib in the differentially sensitive lymphoma cells could provide clues to determine the associated biological mechanisms of actions to ixazomib treatment. Constitutive expression of NFκB has been commonly observed in HL cells including L428 (43). Furthermore, we observed induction of NFκB genes with ixazomib treatment in both ixazomib sensitive L540 and resistant L428 HL cells (Figure 5A), and we subsequently verified NFκB activation occurs in ixazomib-treated multiple TCL and HL cells as shown by western blot assays (data not shown) thus ruling out a significant role for NFκB in resistance to ixazomib. Among the significant upstream regulators affected with ixazomib, inactivation of MYC, which was predicted in Jurkat and L540 cells, were absent in L428. MYC is central to major regulatory pathways including cell cycle, DNA repair, replication, transcription and translation and metabolism (44) with global impact on major “hallmarks of cancer” progression (30). MYC is transcriptionally regulated by cell cycle regulatory proteins (p21, CHK1, CDC2) through chromatin modification via histone acetylation (37) and BRCA1 pathways (45).

We demonstrated that ixazomib affected many of the aforementioned MYC-related biological pathways and expression of Myc protein (Figure 6A). Overexpression of MYC occurs in 30% of all human cancers and frequently predicts for aggressive biological behavior, advanced stage of disease, increased likelihood of relapse, and poor clinical outcome (46, 47). Studies in transgenic mouse models showed that even brief MYC inactivation is sufficient to induce tumor regressions (48) and that targeting MYC may destabilize apoptosis regulatory network and induce cell death in lymphoma (49). In multiple myeloma, a correlation between MYC overexpression (50) and poor response to treatment with bortezomib has been reported, and in mantle cell lymphoma, co-targeting MYC with (CPI203) was shown to overcome bortezomib resistance (51). While these studies show a correlation between MYC and resistance to proteasome inhibition, results from our experiments demonstrated the biological mechanism for MYC dependent resistance to proteasome inhibition, and moreover, how to circumvent these mechanisms to improve therapeutic response.

In L428 cells, poor sensitivity to ixazomib correlated with lack of MYC regulation and histone H3 acetylation. Both histone acetylation and MYC affect similar downstream biological processes including, chromatin modification, gene expression, DNA replication and repair, cell cycle, cytoskeletal function and protein trafficking (52). Chromatin modification involving histone acetylation is mediated by a family of HATs and HDACs with opposing enzymatic activities, and histone acetylation is an important mechanism in the regulation of MYC gene expression (52). Further, the cell cycle regulatory Chk1 is known to regulate histone acetylation via histone H3 phosphorylation, (37) which leads to HDAC3 disassociation favoring MYC transcription (38). Thus in our experiments, CHK1 inhibition alone or in the presence of ixazomib, decreased MYC promoter occupancy (by acetylated Histone H3) and reduced Myc protein in L428 (Figure 7 A&D). Furthermore, by using pharmacologic inhibitors and RNAi for CHK1 and MYC we demonstrated that MYC downregulation was required for inducing cell death with ixazomib. Additionally, CHK1 inhibition reported to enhance cytotoxicity with lenalidomide selectively in MYC-dependent lymphoma (53), support the role for CHK1 dependency in MYC driven tumors. Altogether, results from our analyses showed that ixazomib alone or in combination with CHK1 inhibition have the potential to significantly downregulate MYC and induce potent cell death in TCL and HL models, and it represents a novel combinatorial therapeutic platform for the treatment of these cancers.