Graphical abstract graphic file with name fx1.jpg [29]Open in a new tab Highlights * • Actinomycin D disrupts protein homeostasis in diffuse anaplastic Wilms tumor * • Actinomycin D induces proteasome activity * • Proteasome inhibition sensitizes Wilms tumor cells to actinomycin D * • Higher levels of proteasome components are associated with worse prognosis __________________________________________________________________ Tiburcio et al. investigate drug sensitivity in diffuse anaplastic Wilms tumor, the deadliest type of the most common pediatric renal cancer. They find that actinomycin D, a common chemotherapy, disrupts protein homeostasis and induces sensitivity to proteasome inhibition. Introduction Wilms tumors, or nephroblastomas, are the most common pediatric kidney cancer. Globally, Wilms tumor is diagnosed in 10.4 per 1 million children less than 15 years old each year.[30]^1 North American risk stratification criteria classify Wilms tumors as having favorable or anaplastic histology. The development of effective combinations of chemotherapy, radiation, and surgery has pushed the 5-year overall survival rate to over 90% for those with favorable histology Wilms tumor (FHWT). These strong cure rates have allowed us to de-intensify therapy for some patients with FHWT, where up to 24% of long-term survivors who were treated on historical regimens developed therapy-related chronic health conditions.[31]^2 Other FHWT patients receive intensified therapy based on biological risk factors such as combined loss of heterozygosity (LOH) of chromosomes 1p and 16q or gain of chromosome 1q. On the other hand, patients with diffuse anaplastic Wilms tumor (DAWT), which accounts for ∼10% of Wilms tumor patients, continue to have a relapse rate of over 40% and a 4-year overall survival rate of 66% despite being treated with more aggressive drugs such as doxorubicin, etoposide, cyclophosphamide, and carboplatin.[32]^1^,[33]^3^,[34]^4^,[35]^5 Recent attempts to intensify chemotherapy for those with DAWT have increased short-term toxicity with minimal improvements in cure rate.[36]^6 Anaplastic histology is associated with loss or mutation of p53,[37]^7^,[38]^8 but the molecular mechanisms underpinning chemoresistance in anaplasia are unknown, and there remain no targeted therapies effective in Wilms tumor. Since the 1960s, chemotherapy regimens utilizing actinomycin D (actD), also called dactinomycin, have been routinely used to treat Wilms tumors,[39]^9^,[40]^10 rhabdomyosarcoma, and other sarcomas.[41]^11^,[42]^12 In DAWT, however, actD has been removed from standard protocols. Unlike most FHWT regimens, DAWT patients receive doxorubicin, which can provide higher rates of cure but carries more short- and long-term toxicities, including cardiotoxicity and secondary cancers.[43]^13 Understanding how to improve actD sensitivity without the toxicities associated with anthracyclines like doxorubicin could improve outcomes for Wilms tumor as well as other malignancies. The molecular activity of actD is concentration dependent. At high concentrations, it is a DNA-intercalating agent that can block transcription and DNA replication. However, at the low-nanomolar serum concentrations typically achieved in patients, it primarily inhibits transcription of ribosomal RNA (rRNA), which is transcribed by RNA polymerase I (Pol I), and transfer RNA (tRNA), which is transcribed by RNA polymerase III (Pol III).[44]^14^,[45]^15^,[46]^16^,[47]^17^,[48]^18 Ribosomes are composed of rRNA and ribosomal proteins (RPs), and impaired Pol I activity results in fewer fully formed ribosomes, which consequently reduces global translation. In ribosome-depleted settings, the remaining ribosomes are not uniformly distributed across remaining transcripts; instead, they favor certain transcripts, based on factors such as their intracellular localization, secondary structure, and presence of specific sequence motifs[49]^18^,[50]^19 ([51]Figure 1A). We thus reasoned that the genes preferentially translated following actD exposure could be therapeutically targetable in anaplastic Wilms tumor. To date, however, no published findings have characterized how actD affects translation in Wilms tumors. Figure 1. [52]Figure 1 [53]Open in a new tab ActD disrupts protein homeostasis in anaplastic Wilms tumor cells (A) Diagram of the effect of actD at the translational level. ActD depletes fully formed ribosomes and reduces translational capacity. The reduced ribosomes are distributed unevenly. Ribosome profiling provides a snapshot of transcripts actively undergoing translation. (B) Whole protein lysates of DMSO (vehicle)- or actD-treated WiT49 or 17.94, fluorescently labeled with AHA-AZDye 680 for nascent protein detection, and corresponding loading control (tubulin) detected by western blot from the same gel. (C and D) Top 5 most enriched and bottom 5 most depleted KEGG gene sets in ribosome profiling of actD- vs. DMSO-treated WiT49 after 6 h (C) or 72 h (D). (E) GSEA of genome-wide CRISPR knockout screen reveals top 10 most significant KEGG pathways that sensitize WiT49 to actD, ranked by p value. (F) Heatmap displaying RPPA results of actD- vs. DMSO-treated WiT49. Normalized, log2-transformed, median-centered values from validated antibodies with standard deviation over 0.1 are shown here. In this study, we used ribosome profiling to find that proteasome components are preferentially translated in anaplastic Wilms tumor cell lines following actD treatment. Based on these findings, we studied a combination with the proteasome inhibitor bortezomib (BTZ), and we found that it increases sensitivity of anaplastic Wilms tumor cells to actD in vitro and in vivo. Lastly, DAWTs express higher transcript levels of several proteasome components than relapsed FHWTs (rFHWTs), and higher levels of proteasome components are associated with worse prognosis. Results actD alters the translational landscape of anaplastic Wilms tumor cells actD blocks rRNA transcription at nanomolar doses and mRNA transcription at micromolar doses.[54]^18 To examine the effect of actD on cell viability at nanomolar doses in Wilms tumor, we measured 72-h actD sensitivity in two anaplastic Wilms tumor cell lines, WiT49 and 17.94 ([55]Figure S1A). For these cell lines, we measured half-maximal inhibitory concentration (IC50) to be 1.3 and 2.2 nM, respectively. Next, we confirmed that 2 nM actD reduces levels of 45s pre-rRNA and 18s mature rRNA in both cell lines ([56]Figure S1B). This results in decreased overall protein synthesis, consistent with specific impairment of Pol I activity, as measured by incorporation of both the methionine analog L-azidohomoalanine and O-propargyl-puromycin ([57]Figures 1B, [58]S1C, and S1D). Thus, to understand how actD affects protein levels and cellular functions in Wilms tumor, we next performed three complementary assays in parallel: ribosome profiling, to identify preferentially translated transcripts; reverse-phase protein arrays (RPPAs), to identify differences in protein levels and post-translational modifications; and a CRISPR dropout screen, to identify targetable vulnerabilities. First, to ascertain preferentially translated transcripts, we used ribosome profiling (also known as ribosome footprinting), which provides a snapshot of the transcripts actively undergoing translation.[59]^20^,[60]^21 This technique entails trapping ribosomes on mRNA transcripts, degrading unprotected RNA, and sequencing the RNA fragments that were protected from degradation by ribosomes. Sequencing of the ribosome-protected RNA is computationally compared to RNA sequenced conventionally in the same samples to calculate “translational efficiency,” a measure of the active translation of each transcript. Specifically, we performed ribosome profiling in WiT49 cells treated with actD or vehicle control at 6 h, 72 h, and two weeks. At each time point, we performed ribosome profiling to quantify gene-level differences in translational efficiency in actD-versus vehicle-treated cells. To confirm the expected effect of depleting ribosomes, we examined the locations of the ribosome footprint edges in metagene plots around translation start sites ([61]Figures S2A–S2F). As expected, in both DMSO- and actD-treated cells, there were essentially no detectable reads in 5′ untranslated regions (5′UTRs), while coding sequences exhibit a trinucleotide periodicity, reflecting the reading frame of elongating ribosomes. In DMSO-treated cells, the left edge of ribosome footprints accumulated just upstream of translation start sites, which reflects pausing of the ribosome at the initiation site, as expected for normal conditions ([62]Figures S2A–S2C). In actD-treated cells, however, the initiation site peak was blunted, suggesting that, when ribosomes are depleted, the ribosomes that remain spend less time paused at initiation ([63]Figures S2D–S2F). For all durations of treatment, actD appeared to have minimal effect on the transcriptome, while ribosome footprints revealed gross perturbation of translational landscapes by actD ([64]Figures S3A and S3B). After two weeks of intermittent actD dosing, cells appear to return to a new steady state with globally reduced translation. We next compared the translational efficiency of each gene in actD-treated cells at 6 and 72 h, and we connected preferentially translated genes into Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using gene set enrichment analysis (GSEA) ([65]Tables S1 and [66]S2). The most enriched KEGG pathway by translational efficiency at both time points was KEGG_RIBOSOME, which is composed of RPs and genes that regulate ribosome biosynthesis ([67]Figures 1B, 1C, [68]S4A, and S4B; [69]Tables S3 and [70]S4). This is consistent with reports that RPs are unstable when actD depletes rRNA, which can upregulate translation of RPs via mammalian target of rapamycin complex 1 (mTORC1) signaling.[71]^22^,[72]^23 This increase in translational efficiency was accompanied by a relative decrease in mRNA abundance for RPs ([73]Figure S4C). KEGG_PROTEASOME was the only other gene set in the top five most preferentially translated gene sets at both time points ([74]Figures 1C, 1D, [75]S4D, and S4E; [76]Tables S3 and [77]S4). This increase in translational efficiency was also accompanied by a relative decrease in mRNA abundance for proteasomal proteins ([78]Figure S4F). On the other hand, the most downregulated gene sets at 6 h entailed nucleotide turnover, which led to a depletion of cell-cycle genes at 72 h ([79]Figures 1C and 1D; [80]Tables S3 and [81]S4). In sum, our ribosome profiling of actD-treated cells detected preferential translation of RPs and proteasome components, with a concomitant decrease in translation of cell-cycle genes. Next, we used a genome-wide CRISPR screen to identify therapeutic vulnerabilities in cells with intermittent actD or DMSO for 14 days. We again used KEGG pathways to categorize dependency genes ([82]Figure 1E; [83]Table S5). Here, we again found enrichment for pathways related to protein turnover, including KEGG_PROTEASOME, as well as nucleic acid turnover and cell cycle. Thirdly, since actD regulates protein synthesis, we also performed RPPA on WiT49 cells treated with actD or DMSO for 72 h to understand how actD affects protein levels and post-translational modifications ([84]Figure 1F; [85]Table S6). We found an increase in phosphorylation of some components of the mTORC1 signaling pathway, which mediates the feedback signaling to upregulate translation of RPs in the setting of rRNA depletion.[86]^23 On the other hand, cell-cycle markers such as CDT1, CDC6, and phosphorylated RB1 were depleted in actD-treated cells (also see [87]Figure 3D below). This is consistent with depletion of cell-cycle gene sets in ribosome profiling at 72 h. Together, our three datasets show that, in the presence of actD, Wilms tumor cells choose to preferentially translate ribosome and proteasome components rather than progress through the cell cycle. Figure 3. [88]Figure 3 [89]Open in a new tab BTZ sensitizes anaplastic Wilms tumor cell lines to actD in vitro (A) BTZ kill curves for WiT49 and 17.94 with IC50 values indicated (four-point regression line with shaded region representing 95% confidence interval). Error bars represent mean ± SD from three technical replicates per dose. (B) Heatmaps of Loewe synergy scores for combinations of actD and BTZ in WiT49 and 17.94 cells. (C) Distribution across phases of the cell cycle in WiT49 and 17.94 cells treated with DMSO, actD, BTZ, or combination actD + BTZ for 48 h. (Student’s t test for treated cells versus DMSO: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; G0 and G1 phase shown in green, and S phase in blue). Values shown are mean ± SD from two technical replicates. (D) Effect of 48-h treatment WiT49 and 17.94 cells with DMSO, actD, BTZ, and combination actD + BTZ on levels of cell-cycle and apoptosis markers by western blot. mTORC1 inhibition does not sensitize Wilms tumor cells to actD Based on phosphorylation of mTORC1 signaling intermediates in RPPA results and prior interest in mTORC1 signaling in Wilms tumor,[90]^24^,[91]^25 we next investigated how actD affected mTORC1 signaling in WiT49 and a second anaplastic Wilms tumor cell line, 17.94. Using Western blots, we confirmed that actD induced phosphorylation of AKT and 4E-BP1 in WiT49 and 17.94 cells ([92]Figures S4G and S4H) (The phosphorylation of another mTORC1 target, p70S6K, was not clearly upregulated by actD). We treated WiT49 and 17.94 with rapamycin at doses up to 100 μM and found that this drug confers a more cytostatic rather than cytotoxic effect ([93]Figure S4I). We also measured cell viability in combinations of actD and the mTORC1 inhibitor rapamycin using the Loewe independence model ([94]Figure S4J; [95]Tables S7 and [96]S8). However, the interaction did not consistently show synergy, as we only observed synergy above the upper limit of serum concentrations typically achieved in patients (∼2–3 nM for actD[97]^14 and 16 nM for rapamycin[98]^26). In other words, mTORC1 inhibition with rapamycin did not appear to consistently sensitize Wilms tumor cells to actD, and rapamycin alone was not cytotoxic, even at very high doses. ActD induces proteotoxic stress and is synergistic with proteasome inhibition in anaplastic Wilms tumor cells One effect of mTORC1 signaling is to upregulate translation of RPs,[99]^27 and KEGG_RIBOSOME was the most enriched gene set in actD-treated cells. However, we found that 72-h actD treatment in fact appeared to slightly reduce the total protein levels of multiple RPs in both WiT49 and 17.94 ([100]Figures S5A and S5B). This could be because excess RP subunits are unstable without rRNA and are degraded by the proteasome to maintain appropriate RP:rRNA stoichiometry.[101]^22^,[102]^28^,[103]^29 While we did not observe an increase in RP levels with actD treatment, we did observe accumulation of proteasome components. When we exposed WiT49 and 17.94 to actD for up to 72 h, both cell lines exhibited an appreciable increase in several proteasome complex subunits at one or more time points between 18 and 72 h ([104]Figures 2A and 2B). We specifically measured PSMD1 (also known as P112), PSMA6 (subunit α1), PSMB1 (β6), PSMB2 (β4), and PSMB5 (β5), as well as the molecular chaperone for proteasome assembly, the proteasome maturation protein (POMP). In both cell lines, PSMA6, PSMD1, and POMP peaked at 18–24 h. PSMB1 peaked at 72 h in WiT49 but rose earlier in 17.94 and remained high, while PSMB2 peaked at 24 h in 17.94 but rose earlier in WiT49 and remained high. Lastly, PSMB5 peaked at 48–72 h in WiT49 but did not appear to rise in 17.94 at the time points we examined. These increases in protein levels of proteasome components corresponded to increased proteasome chymotrypsin-like enzymatic activity in both cell lines, which was blunted by co-treatment with the proteasome inhibitor BTZ ([105]Figure 2C). Figure 2. [106]Figure 2 [107]Open in a new tab ActD promotes proteasome level and activity in anaplastic Wilms tumor cells (A and B) Western blots for (A) and quantification of (B) PSMA6 (α1), PSMB1 (β6), PSMB2 (β4), PSMB5 (β5), PSMD1 (P112), and POMP in WiT49 and 17.94 following 18-, 24-, 48-, and 72-h 2 nM actD treatment versus vehicle (DMSO). (C) Relative proteasome-specific chymotrypsin-like activity in WiT49 and 17.94 cells following 24 h of DMSO versus 2 nM actD, 8 nM BTZ, or 2 nM actD + 8 nM BTZ treatments (Student’s t test p value versus vehicle: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). Error bars represent mean ± SD from four technical replicates per condition. Because actD caused preferential translation of proteasome subunits, we next examined how WiT49 and 17.94 respond to proteasome inhibition. These two cell lines were sensitive to BTZ alone at nanomolar concentrations, in the range of serum levels typically achieved in patients[108]^30^,[109]^31 ([110]Figure 3A). This is close to the average sensitivity (3.9 nM) observed across the National Cancer Institute 60 (NCI-60) cell lines screen.[111]^32 Furthermore, at or below these levels, actD and BTZ synergistically inhibited WiT49 and 17.94 to different degrees ([112]Figure 3B; [113]Tables S9 and [114]S10). In WiT49, actD and BTZ acted synergistically across nearly all combination concentrations. Loewe scores in 17.94 showed synergy at lower concentrations and additivity at medium and higher concentrations. Taken together, these data suggest that the addition of BTZ could be a strategy to enhance or restore actD sensitivity in anaplastic Wilms tumor cells. Our RPPA results ([115]Figure 1F) had shown a reduction in cell-cycle markers in actD-treated WiT49, so we next examined how actD and BTZ affect cell-cycle progression using flow cytometry ([116]Figure 3C). Specifically, we treated WiT49 and 17.94 with actD and/or BTZ for 48 h, and we measured the proportion of cells in each phase of the cell cycle using flow cytometry ([117]Figure 3C). We found that actD increased the proportion of cells in G0/G1 and reduced the proportion in the S phase in both cell lines, though the difference was only statistically significant in WiT49. This is consistent with previous reports that low-dose actD treatment leads to G1 pause.[118]^33^,[119]^34 In contrast, BTZ increased the proportion of cells in G2 and reduced the proportion in the S phase, consistent with previous reports that BTZ causes cells to accumulate in G2/M phase.[120]^32 BTZ has previously been shown to trigger cell death, cell-cycle arrest, and autophagy in cancer cell lines,[121]^35^,[122]^36^,[123]^37^,[124]^38^,[125]^39^,[126]^40 so we next measured cell-cycle and apoptosis markers using western blots. In both cell lines, actD alone reduces cell-cycle markers, including chromatin licensing and DNA replication factor 1 (CDT1), cyclin D2, cyclin E1, and cyclin A2 ([127]Figure 3D).[128]^41^,[129]^42^,[130]^43 These cell-cycle regulators are synthesized and degraded in each turn of the cell cycle.[131]^44^,[132]^45 Consistent with our flow cytometry results, these cell-cycle markers were more affected by actD in WiT49 than 17.94. These support our findings from CRISPR screen and protein arrays, which showed that actD induced a dependency on cell-cycle genes and led to a fall in cell-cycle markers ([133]Figures 1E and 1F). In addition to impaired cell-cycle progression, use of the proteasome inhibitor BTZ resulted in the accumulation of apoptotic markers in both cell lines ([134]Figure 3D).[135]^46^,[136]^47^,[137]^48^,[138]^49 ActD alone induced a slight increase in cleaved caspase-7 and cleaved poly(ADP-ribose) polymerase (PARP) in 17.94 cells, but both apoptosis markers were induced by BTZ in both cell lines. The combination of these effects supports the potential for using actD and BTZ to target anaplastic Wilms tumors. Combined treatment of actD and BTZ in Wilms tumor xenografts suppresses growth in vivo We next examined the effect of combining BTZ with actD against two anaplastic Wilms tumor lines in vivo: cell line-derived xenografts from 17.94 and the TP53-mutated patient-derived xenograft (PDX) line KT-53.[139]^50^,[140]^51 We implanted both lines into immunocompromised NOD scid gamma (NSG) mice and treated them with vehicle only, actD, BTZ, or both. We found that combination treatment significantly reduced tumor volume and conferred a significant survival advantage for mice bearing KT-53 xenografts compared to the other three arms ([141]Figures 4A and 4B). Similarly, 17.94 xenografts treated with the combination of actD and BTZ were significantly smaller than those who received vehicle control or BTZ alone ([142]Figures 4C and 4D) (Although it did not reach statistical significance, tumor volume in the combination treatment cohort was also slightly smaller than the actD-only arm). In these treatments, BTZ was well tolerated and did not appear to add toxicity. There was no difference in body weight between mice receiving BTZ and vehicle ([143]Figure S6A and S6B). Similarly, the weights of mice treated with the combination of actD and BTZ were similar to the weights of mice treated with actD alone. Figure 4. [144]Figure 4 [145]Open in a new tab BTZ sensitizes subcutaneous anaplastic Wilms tumor xenografts to actD (A) Subcutaneous tumor volumes of NSG mice bearing KT-53 xenografts treated with vehicle, actD, BTZ, and combination actD + BTZ (Student’s t test of endpoint volumes of combination versus vehicle or actD only: ∗p < 0.05). (B) Kaplan-Meier survival curves for KT-53 tumor-bearing mice treated with vehicle, actD only, BTZ only, and combination (log rank test versus vehicle, ∗∗p < 0.01). (C) Subcutaneous tumor volumes of NSG mice bearing 17.94 xenografts treated with vehicle, actD only, BTZ only, and combination (Student’s t test of endpoint volumes of combination versus vehicle: ∗∗∗p < 0.001). (D) Kaplan-Meier survival curves for 17.94 tumor-bearing mice treated with vehicle, actD only, BTZ only, and combination. (E and F) Quantification of Ki-67-positive nuclei (E) and TUNEL-positive nuclei (F) in KT-53 and 17.94 subcutaneous tumors treated with vehicle, actD only, BTZ only, or combination actD + BTZ. Values shown are mean ± SD, calculated from two tumors each with four fields of view for all treatment conditions (Student’s t test p value versus vehicle unless otherwise indicated: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). Based on the effects of actD and BTZ on cell cycle and apoptosis we had observed in vitro, we next measured cell-cycle and apoptosis markers in these subcutaneous tumors. We used immunohistochemistry for Ki-67 as a marker of proliferation and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay as a marker of apoptosis. In both lines, tumors from combination-treated mice had a statistically significant reduction in proliferation compared to vehicle or BTZ alone ([146]Figures 4E and [147]S7A–S7D). Similarly, in both lines, combination treatment yielded significantly more apoptosis than vehicle and actD alone ([148]Figures 4F, [149]S7E, and S7F). Taking these into consideration, we find that the compounding effects of adding BTZ to actD could be a powerful approach to targeting anaplastic Wilms tumor cells. Proteasome subunit expression levels correlate with outcome Lastly, we examined expression of proteasome genes in publicly available RNA sequencing (RNA-seq) data from 42 DAWT and 83 rFHWT samples generated by the National Cancer Institute Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project.[150]^8 Compared to rFHWT, the single most enriched KEGG gene set in DAWT was KEGG_PROTEASOME ([151]Figures 5A and [152]S8A; [153]Table S11). Similarly, two of the three most enriched Reactome gene sets were related to anaphase-promoting complex (APC/C)-mediated degradation of cell-cycle proteins, suggesting that proteasomal degradation could promote proliferation in DAWT by degrading proteins in each cell cycle ([154]Figures 5B and [155]S8B; [156]Table S12). Figure 5. [157]Figure 5 [158]Open in a new tab RNA expression of proteasome genes in TARGET anaplastic histology and relapsed favorable histology Wilms tumors (A) Top KEGG pathways enriched in RNA-seq of DAWT versus rFHWT. (B) Top Reactome pathways enriched in RNA-seq of DAWT versus rFHWT. (C and D) Overall survival of Wilms tumor patients stratified according to the expression of proteasome enzymatic subunit genes PSMB1, PSMB2, and PSMB5 among patients with relapsed favorable histology tumors (C) or anaplastic histology tumors (D) (log rank test p value: ∗p < 0.05, ∗∗p < 0.01). Next, we examined whether expression of proteasome subunits correlated with outcome in Wilms tumor. We stratified rFHWT and DAWT patients into three categories based on high, medium, or low expression of the enzymatic proteasome subunits PSMB5 (β5), PSMB6 (β1), and PSMB7 (β2) based on expression Z scores for each gene. Tumors with at least one gene Z score ≥ +1 were categorized as “high,” while those with at least one gene Z score ≤ −1 were categorized as “low.” Samples with Z scores between −1 and +1 for all three genes were categorized as “medium,” while those with one gene Z score ≥ +1 and another gene Z score ≤ −1 were omitted. Among rFHWT patients, proteasome-high patients fared far worse than proteasome-low or proteasome-medium patients ([159]Figures 5C and [160]S9A). However, this is a clinically heterogeneous population comprising patients who would have received different treatments based on clinical stage, chromosomal LOH, and other factors. Thus, we next examined whether proteasome expression correlated with survival within each clinical stage. To increase statistical power, we combined the proteasome-low and proteasome-medium groups, which fared similarly to each other ([161]Figure 5C). We then compared the survival of the proteasome-high group to the combined proteasome-low and proteasome-medium group within each stage ([162]Figures S9B–S9E). We found that high proteasome gene expression still correlated with significantly worse outcome in stages II and IV (p = 0.007 and 0.002, respectively). Although the correlation was not significant for the other groups, these analyses were limited by small sample sizes after subdividing by stage and proteasome expression. There was only one proteasome-high patient in the stage I rFHWT cohort. In the stage III rFHWT cohort, proteasome-high patients fared slightly worse but did not reach statistical significance (p = 0.15). Among DAWT patients, who do not usually receive actD-based therapy regimens, the relationship between proteasome expression and outcome was less evident; proteasome-medium patients fared worse than proteasome-low patients, but proteasome-high patients were not significantly different from proteasome-medium or proteasome-low[163]^8^,[164]^52^,[165]^53 patients ([166]Figure 5D). Together, these data suggest that proteasome subunit levels may underlie some of the clinical differences between DAWT and FHWT and that high proteasome subunit levels correlate with poorer prognosis in rFHWT. Proteasome levels could be prognostic in some subgroups, and proteasome inhibition could benefit some of these patients. Discussion Developments in Wilms tumor research have illuminated the mutational, epigenetic, mRNA, and microRNA expression landscapes of these tumors, yet relevant targetable vulnerabilities remain elusive.[167]^8^,[168]^54^,[169]^55^,[170]^56^,[171]^57^,[172]^58 Anaplastic Wilms tumors exhibit relative resistance to conventional chemotherapy, including actD, and little is known about how to overcome such resistance. Furthermore, despite its widespread use, little is known about how actD influences the translational landscape of cancer. Defining the mechanisms underlying these effects and their consequences could potentially uncover targetable vulnerabilities in relatively chemo-refractory anaplastic Wilms tumors, which could enhance outcomes while minimizing off-target toxicities in survivors. Through several orthogonal approaches, our work reveals that actD disrupts protein homeostasis in Wilms tumor and suggests proteasome inhibition as a potential targeted therapy for inducing actD sensitivity in anaplastic Wilms tumors. Protein homeostasis involves a synchronized and responsive balance between protein synthesis and degradation, which are both affected by actD. Since protein synthesis is energy intensive, particularly in rapidly proliferating cancers where proteins like cyclins are continuously synthesized and degraded, maintaining protein homeostasis is crucial to ensure optimal levels of amino acids and other nutrients for growth.[173]^59^,[174]^60^,[175]^61 Blocking protein synthesis with actD leads cells to upregulate Akt/mTORC1 signaling, which can increase the translation of RP genes.[176]^27^,[177]^62^,[178]^63^,[179]^64 Although we detected an increase in translational efficiency of RP genes, they did not accumulate by western blot in our 72-h treatments, possibly due to their instability without rRNA. Although the mTOR pathway is a potential therapeutic target in Wilms tumor and other cancers,[180]^65^,[181]^66^,[182]^67 the combination of actD and rapamycin did not consistently show synergy in vitro for either cell line, and rapamycin alone only exhibited a cytostatic effect. Our cells were in nutrient-rich media, which may be compounding to the variables that dictate the effect of rapamycin,[183]^66^,[184]^68 and our results do not rule out the possibility that other mTOR signaling inhibitors could still have potential. The importance of protein homeostasis in Wilms tumor is also supported by other recent findings. Common Wilms tumor mutations interfere with protein homeostasis. For instance, recurrent mutations in CTNNB1 and MYCN prolong their stability by interfering with their degradation by the proteasome.[185]^8^,[186]^69 Other common mutations impair processing of microRNAs, which normally regulate translation of target transcripts.[187]^54^,[188]^57^,[189]^58^,[190]^70 Recently, a small-molecule inhibitor of histone lysine demethylases KDM4A-C was found to act by reducing rRNA and RP transcripts in WiT49 cells, which led to a broad reduction in protein translation.[191]^71 How actD or BTZ interacts with common Wilms tumor mutations or with KDM4A-C inhibition remains to be seen. Based on our finding that proteasome subunits were preferentially translated after actD exposure, we found that proteasome inhibition with BTZ sensitizes cells to actD in vitro and in vivo. The proteasome is upregulated in response to proteotoxic stresses,[192]^28^,[193]^29^,[194]^72 and Akt/mTORC1 activates proteasome subunit expression via Nrf1/NFE2L1[195]^73^,[196]^74^,[197]^75^,[198]^76 to enhance the intracellular amino acid pool and the unfolded protein response. BTZ was the first proteasome inhibitor approved by the United States Food and Drug Administration,[199]^77^,[200]^78^,[201]^79 and it has been tested in pediatric cancer patients.[202]^30^,[203]^80^,[204]^81 However, other than a study showing a lack of cross-resistance between actD and BTZ in gliobastoma,[205]^82 no published studies have explored combinatorial treatment of actD and BTZ to our knowledge. While proteasome inhibitors have been clinically successful in hematological cancers, single-agent proteasome inhibitor trials have not been as successful for solid tumors.[206]^83^,[207]^84 It is thought that this could be due to insufficient pharmacokinetic distribution of BTZ. Strategies for enhancing the effect of BTZ in solid tumors include liposomal nanoformulations[208]^85 and combination with inhibiting other proteasomal components.[209]^86 Our data suggest that actD could be another way to enhance the effect of BTZ in solid tumors. While we did not observe increased toxicity with this approach in animals, it remains to be seen whether this combination would be tolerated in patients. Use of newer proteasome inhibitors, such as carfilzomib, ixazomib, marizomib, and oprozomib, can also be considered.[210]^87^,[211]^88 Compellingly, for our xenograft studies in both KT-53 and 17.94, systemic combination treatment reduced proliferative cells and increased apoptosis. While our in vitro experiments test sensitivity over a 72-h period, in vivo experiments more closely model the once-weekly actD dosing used in patients.[212]^89 Our study on KT-53 recapitulated a previously demonstrated insensitivity to actD,[213]^51 which we found could be overcome by BTZ. For 17.94, which was sensitive to nanomolar actD concentrations in vitro, actD alone was also effective in vivo, and adding BTZ produced a small additional effect. The different effects we observed in sensitivity to actD and BTZ suggest that other factors regulating protein homeostasis may also contribute to how cells respond to these drugs. Further studies involving more cell lines, other cancer types, or manipulation of individual factors that regulate protein homeostasis might help explain these differential responses. Together, our findings indicate that the combined use of actD and BTZ could be a promising strategy for combating therapy resistance in anaplastic Wilms tumors. We demonstrated that actD impairs cell-cycle progression in vitro and in vivo, which we attribute to dysregulated proteostasis.[214]^15^,[215]^90^,[216]^91 These effects were more pronounced in WiT49 than 17.94. Normally, D-type cyclin levels accumulate in response to mitogenic signaling, promoting the transcription of S-phase genes such as E-type cyclins and chromatin licensing and DNA replication factor 1 (CDT1).[217]^44^,[218]^92^,[219]^93 Consistent with our RPPA in WIT49, we observed in both cell lines that actD reduces cyclin D2, cyclin E1, and CDT1 levels, suggesting that actD prevents transition to S-phase, when cyclin A is produced. On the other hand, these proteins are normally targeted for proteasomal destruction by the SKP1-CUL1-F-boc protein (SCF) ubiquitin ligase,[220]^94 and, indeed, we found that BTZ caused them to accumulate. Moreover, BTZ treatment induced apoptosis in vitro and in vivo, consistent with studies on other cancer cell lines.[221]^35^,[222]^36^,[223]^37^,[224]^38^,[225]^39^,[226]^40 In the TARGET cohort,[227]^8 we found that higher levels of proteasome gene expression correlate with anaplastic histology and with poor outcome among some subgroups of rFHWTs. Studies across other types of cancer show that the association of proteasome activity with prognosis is context specific.[228]^95 Lower expression of proteasome subunit genes was associated with reduced survival in head and neck squamous cell carcinoma,[229]^96 while studies in breast cancer, glioma, and hepatocellular carcinoma found that higher expression of proteasome subunit genes is associated with worse survival.[230]^97^,[231]^98^,[232]^99 Here, we correlate proteasome expression with worse outcome in Wilms tumor. Anaplastic Wilms tumors are strongly associated with TP53 mutation,[233]^7^,[234]^8 and TP53 mutation is known to contribute to proteasome subunit overexpression.[235]^100 To our knowledge, this is the first study to explore the translational landscape of actD treatment in Wilms tumor. Through in vitro and in vivo models, we propose a model of protein homeostasis-dependent actD sensitivity ([236]Figure S10). Use of actD impairs ribosome biogenesis and cell-cycle progression while upregulating proteasome activity. Higher proteasome capacity may help Wilms tumor cells escape front-line chemotherapeutics like actD, and this effect may be reversed with proteasome inhibition. Our complementary approaches converged on proteasome activity as a potential mechanism for chemoresistance. Repurposing widely used drugs like BTZ could allow for an accelerated path to clinical translation. More broadly, this strategy could improve treatment not only for Wilms tumor but also for other tumors where actD is used, such as rhabdomyosarcoma and Ewing sarcoma. Limitations of the study Our conclusions are limited by the fact that our in vitro studies focused on two anaplastic Wilms tumor cell lines, and our in vivo studies focused on two anaplastic Wilms tumor PDX lines. With such a small sample size, we cannot examine whether other correlates, such as patient demographics or sex, may affect the generalizability of our conclusions. We did not compare them to FHWT cell lines or xenografts, and we cannot know how our observations might extend to non-anaplastic Wilms tumors. Similarly, our analysis of human Wilms tumor RNA-seq was based on published TARGET data, which only include rFHWT and DAWT. As such, we do not know whether expression of proteasome components would predict outcome in a population of FHWT at diagnosis. Similarly, as these are retrospective data, the predictive power of these expression patterns needs to be validated in an independent cohort. Lastly, just as in vitro findings may vary from in vivo results, pre-clinical models cannot replace clinical trials. To our knowledge, actD and BTZ have not been given in combination, and the toxicities of such a regimen are unknown. Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Kenneth S. Chen (kenneth.chen@utsouthwestern.edu). Materials availability No new unique reagents were generated in this study. Data and code availability This paper does not report original code. Ribosome profiling and accompanying RNA-seq data from WiT49 are deposited in the NCBI GEO database under accession number GEO: [237]GSE270330 . Any additional information required to reanalyze the data reported in this work is available from the [238]lead contact upon request. Acknowledgments