Introduction
Acute myeloid leukemia (AML) constitutes a clonal hematopoietic stem cell malignancy characterized by the accumulation of immature progenitor cells with compromised differentiation, leading to the impairment of hematopoietic function [ 1 , 2 ]. Approximately 20% of individuals diagnosed with AML exhibit leukocytosis, with peripheral blood leukocyte counts exceeding 50 × 10^9/L or 100 × 10^9/L. This group is referred to as hyperleukocytic AML (HL‐AML) [ 3 , 4 ]. HL‐AML emerges as an oncological emergency due to its elevated risk of early mortality resulting from leukemic infiltration of organs, leukocyte stasis syndrome, diffuse intravascular coagulopathy, and tumor lysis syndrome. HL‐AML manifests with an abrupt onset, rapid progression, relatively low remission rate, and a notably high early mortality rate, reaching up to 40% within 1 week [ 5 , 6 , 7 ]. It is classified as a high‐risk subtype of acute leukemia. Due to its severe early lethality, certain patients with HL‐AML face challenges in undergoing conventional treatment or novel drug therapy promptly. HL‐AML displays significant heterogeneity in terms of morphology, immunophenotype, cytogenetic and epigenetic features, as well as response to therapy [ 4 , 8 , 9 ]. Therefore, investigating the characteristics and potential therapeutic targets of HL‐AML patients assumes paramount importance.
Serum proteins play dual roles as contributors to diseases and potential therapeutic targets. Proteomic analysis stands as a crucial method for identifying biomarkers in biological and biomedical samples [ 10 , 11 , 12 ]. This technique entails the exhaustive characterization of the proteome of a cell or biofluid, facilitating the identification and subsequent bioinformatics analysis of proteins exhibiting differential expression. Serum‐based proteomics, in particular, offers an advantage in unveiling the progression of diseases. An increasing body of research indicates that combining transcriptomics and proteomics technologies to analyze the complex molecular mechanisms of hematologic malignancies is a promising approach. However, specific serum proteomic analysis of HL‐AML has yet to be reported.
Here, we conducted a label‐free proteomic analysis of serum from patients with HL‐AML and NHL‐AML, integrating the results with publicly available transcriptomic data. The findings unveiled a pronounced enrichment of the NF‐kappa B pathway in HL‐AML. Additionally, bortezomib played a pivotal role in the treatment of HL‐AML patients. This research offers unprecedented insights into the molecular distinctions between HL‐AML and NHL‐AML, deepening our comprehension of their mechanisms at the molecular level and providing guidance for refining therapeutic strategies against this malignancy.
Materials and Methods
Patient Population
Participants were recruited from Wuhan University Zhongnan Hospital in Wuhan, China, diagnosed and classified as AML based on the 2016 World Health Organization criteria. The study included newly diagnosed AML patients. These patients are stratified into HL‐AML and NHL‐AML categories based on whether their white blood cell (WBC) counts exceed 50 × 10^9/L. Informed consent was obtained, and ethical approval was secured from Zhongnan Hospital of Wuhan University. Detailed patient information is provided in Table S1 .
Proteomics Analysis
Label‐free LC–MS/MS quantitative proteomic analysis was conducted by Jingjie PTM BioLab. Bone marrow samples were collected from four specimens from patients with HL‐AML and four specimens from patients with NHL‐AML, followed by centrifugation at 3000 rpm for 10 min. The serum was then stored at −80°C until further use. After thawing at −80°C, the samples were centrifuged at 4°C for 10 min at 12,000 g to remove cellular debris. The supernatant was transferred into fresh centrifuge tubes, and high‐abundance proteins were depleted according to the manufacturer's protocol (Pierce Top 14 Abundant Protein Depletion Spin Columns Kit, Thermo Scientific). Protein concentration was determined using the BCA kit. An aliquot of the protein from each sample was used for enzymatic digestion. Peptides were solubilized in mobile phase A and separated via an EASY‐nLC 1200 ultra‐performance liquid chromatography system (UPLC). Mobile phase A consisted of an aqueous solution containing 0.1% formic acid and 2% acetonitrile, while mobile phase B comprised 0.1% formic acid and 90% acetonitrile. The liquid‐phase gradient settings were: 0–68 min, 4%–20% B; 68–82 min, 20%–32% B; 82–86 min, 32%–80% B; and 86–90 min, 80% B. The flow rate was maintained at 500 nL/min. Peptide fragments were separated via the UPLC system, ionized using the NSI Ion Source, and subsequently analyzed using the Orbitrap Exploris 480 mass spectrometer. Protein sequences were identified via Proteome Discoverer (v2.4.1.15) software, employing Homo_sapiens_9606_PR_20210721.fasta database (78,120 sequences). The false discovery rate (FDR) for protein, peptide, and PSM identification were all set to 1%. Significantly different proteins ( p < 0.05) underwent subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.
Data Collection
Gene expression profiles and clinical parameters were obtained from cBioPortal. Differentially expressed genes (DEGs) were identified using the limma package in R, with criteria of adjusted p < 0.05, and |log2FC| > 0.5. Functional annotation utilized GO and KEGG pathway through the “ClusterProfiler” R package.
Cell Counting, Apoptosis, and Cell Proliferation
Cell counting was conducted utilizing trypan blue cell counting. For apoptosis assessment, the Annexin V‐FITC/PI apoptosis assay kit (Lianke Biotechnology) was employed in accordance with the manufacturer's instructions. BrdU staining was performed following the manufacturer's protocol using the BD BrdU Flow Kit.
Animal Experimentation
NSG mice (female, 5 weeks old) were procured from Jiangsu Jicui Biotechnology Co. Ltd. Bone marrow blood from HL‐AML patients was subjected to gradient centrifugation to isolate mononuclear cells, which is instrumental in model construction. Each mouse was intravenously injected with 1 × 10^7 HL‐AML cells to induce leukemia. Successful establishment of a PDX HL‐AML model was confirmed upon the observation of HL‐AML cells in mouse bone marrow smears. Subsequently, the mice were randomly allocated into four groups, with a 1‐week dosing regimen. The DA group received daily intraperitoneal injections of cytarabine (30 mg/kg) for 7 consecutive days. Additionally, on Days 1, 3, and 5, intraperitoneal injections of daunorubicin (1.5 mg/kg) were administered. The bortezomib (1 mg/kg) group received intraperitoneal injections of bortezomib on Days 1, 2, 4, and 6. The DA combined with the bortezomib group underwent 7 consecutive days of intraperitoneal cytarabine administration, daunorubicin injections on Days 1, 3, and 5, and bortezomib injections on Days 1, 2, 4, and 6. The control group received equivalent volumes of physiological saline with matching frequency. At the end of the experiment, mice were executed and tissues were harvested for further study. Information on flow cytometry antibodies is provided in Table S2 .
Statistical Analysis
Statistical analyses were performed using GraphPad Prism (version 8.0) or R script. In survival experiments, mouse survival rates were analyzed using the log‐rank test. Differences between groups were analyzed using two‐tailed t ‐tests. Data are presented as mean ± standard deviation (SD).* p < 0.05, ** p < 0.01, *** p < 0.001.
Results
Serum Proteomic Analysis of HL ‐ AML Patients
AML patients were stratified into high leukocytosis (WBC > 50 × 10^9/L) and non‐high leukocytosis (WBC< 50 × 10^9/L) based on their WBC count. Label‐free proteomic analysis of bone marrow serum samples from four cases of HL‐AML patients and four cases of NHL‐AML patients was conducted (Figure 1A ). Following quality control standardization of the dataset, a total of 1297 serum proteins were detected. Principal component analysis (PCA) was initially employed to delineate the relationship between HL‐AML and NHL‐AML; serum proteomics distinctly differentiated HL‐AML samples from NHL‐AML samples, as illustrated in Figure 1B . To elucidate the enriched proteins in the serum of HL‐AML patients, a comparative analysis of the quality‐controlled 1297 serum proteins was undertaken, identifying 160 differentially expressed serum proteins ( p < 0.05). Among these, 71 proteins were upregulated, while 89 proteins were downregulated (Figure 1C and Table S3 ). Enrichment analysis was performed to explore the biological functions of these differentially expressed proteins. GO analysis revealed biological processes (BP) encompassing “blood coagulation” and “hemostasis,” while the most abundant terms in cellular components (CC) were “secretory granule lumen” and “cytoplasmic vesicle lumen,” and in molecular functions (MF), it was “integrin binding” (Figure 1D and Table S4 ). Furthermore, KEGG pathway enrichment analysis indicated significant enrichment in classical tumor‐related pathways such as the PI3K‐Akt signaling pathway, leukocyte transendothelial migration, and NF‐kappa B signaling pathway ( q < 0.05, Figure 1E ). To further elucidate the functions of the differentially expressed proteins, we performed KEGG pathway analysis using ClueGO (version 2.5.7) ( https://apps.cytoscape.org/apps/cluego ). The ClueGO enrichment analysis indicated that the differentially expressed proteins were primarily involved in the complement and coagulation cascades, NF‐kappa B signaling pathway, and leukocyte transendothelial migration ( p < 0.05, Figure 1F ).
The NF ‐Kappa B Pathway as a Candidate Therapeutic Target for HL ‐ AML
In the OHSU‐AML cohort, we identified 1211 DEGs (adjusted p < 0.05, and |log2FC| > 0.5). Among these, 387 genes were upregulated, and 824 genes were downregulated. The volcano plot is presented in Figure 2A . GO analysis revealed the enrichment of these DEGs in positive regulation of cell activation, external side of the plasma membrane, and antigen binding (Figure 2B and Table S5 ). KEGG results elucidated potential biological functions, encompassing a total of 18 significantly enriched pathways across environmental information processing, organismal systems, and human diseases ( q < 0.05, Figure 2C ). By intersecting the enriched pathways from the proteomic and transcriptomic analyses, we observed enrichment of the NF‐kappa B pathway at both the gene and protein levels. Given this convergence, we subsequently focus on unraveling the potential significance of the NF‐kappa B pathway in HL‐AML patients (Figure 2D ).
Inhibition of HL ‐ AML Cell Proliferation by Bortezomib In Vitro
Following the notable enrichment of the NF‐kappa B pathway in HL‐AML, we aimed to assess whether its inhibition could represent an effective therapeutic strategy. Bortezomib, a proteasome inhibitor with well‐known properties of inhibiting the NF‐kappa B pathway, has been approved for the treatment of a variety of hematologic malignancies [ 13 , 14 ]. To this end, we collected bone marrow cells from three HL‐AML patients and treated HL‐AML cells with increasing concentrations of bortezomib (0–20 nM) to examine the killing effect of bortezomib on HL‐AML cells (Figure 3A ). The results showed a significant decrease in viable HL‐AML cells and a notable increase in apoptosis rates (Figure 3B–D ). Further, BrdU assays assessing cell proliferation reveal a marked inhibition of HL‐AML cell proliferative capacity by bortezomib (Figure 3E,F ). Additionally, we compared the cytotoxic effects of bortezomib on HL‐AML versus NHL‐AML. Bone marrow cells were collected from three patients with HL‐AML and three with NHL‐AML, followed by isolation of mononuclear cells using density gradient centrifugation. After treating the cells with bortezomib for 48 h, flow cytometry was used to assess apoptosis, revealing that bortezomib exerted a stronger cytotoxic effect on HL‐AML compared to NHL‐AML (Figure S1 ). These results indicate the cytotoxicity of bortezomib on HL‐AML cells.
Bortezomib Combined With DA Regimen Suppresses HL ‐ AML Growth In Vivo
In the clinical domain of AML therapeutics, the “3 + 7” chemotherapy regimen, epitomized by the combination of cytarabine and daunorubicin in the DA protocol, has become the standard of induction therapy in this pathology. To ascertain the potential augmentation of efficacy, we probed the synergy between bortezomib and the DA regimen within a constructed PDX model of HL‐AML. NSG mice were intravenously infused with cells derived from HL‐AML patient, subsequently stratified into four cohorts for systematic drug administration (Figure 4A ). Treatment with bortezomib in combination with the DA regimen provided a significant survival advantage compared to the other three groups. (Figure 4B ). Samples were taken 1 week after drug administration, and a significant reduction of HL‐AML cells was observed in the bortezomib combined with the DA regimen group compared with the other three groups by hematoxylin and eosin staining on bone marrow smears (Figure 4C ). Concurrently, discernible reductions in CD45 + and CD45 + CD33 + ratios, along with absolute cell count ratios, were documented in the bortezomib‐combined DA regimen group, indicative of disease remission (Figure 4D–I ). These findings posit that the amalgamated therapeutic approach involving bortezomib and the DA regimen exerts a profound and discernible influence on the therapeutic intervention of HL‐AML.
Bortezomib Enhances Therapeutic Efficacy by Suppressing the NF ‐Kappa B Pathway
To validate the mechanism of bortezomib, we examined key molecules on the NF‐kappa B pathway, namely, P‐IKBα and P‐p65. The results of bone marrow immunohistochemistry revealed a significant reduction in CD33, P‐IKBα, and P‐p65 in mice treated with the combination of bortezomib and DA regimen (Figure 5A ). Therefore, bortezomib enhances the anti‐tumor effect on HL‐AML by suppressing the NF‐kappa B pathway.
Discussion
Over the past three decades, remarkable advancements have been achieved in the therapeutic landscape of AML, primarily attributable to the introduction of treatment regimens based on anthracycline and cytarabine [ 15 , 16 , 17 ]. However, a significant proportion of patients with AML experience relapse or fail to respond to standard therapy. HL‐AML is a high‐risk type of leukemia with a peripheral blood leukocyte count of 50 × 10^9/L or 100 × 10^9/L. It has an acute onset, is prone to early complications, and, without intervention, can have an early mortality rate of 20% to 40%, requiring urgent treatment. Despite the development of therapeutic modalities, including hydroxyurea, low‐dose chemotherapy, and leukapheresis, current evidence suggests that tailored supportive care has yet to meaningfully impact early mortality rates in HL‐AML patients [ 18 , 19 , 20 ]. A comprehensive understanding of the pathogenic mechanisms governing HL‐AML is crucial to circumvent its occurrence and optimize prognostic outcomes. With the application of high‐throughput molecular biology techniques such as transcriptomics and proteomics, researchers have delved into the intricate BP underpinning cancer. A more exhaustive exploration into the pathogenesis of HL‐AML sets the stage for deeper investigation.
In this study, bone marrow serum from HL‐AML patients was analyzed proteomically using label‐free proteomics. Through comprehensive bioinformatics analyses, we unveiled disparities in protein functionality and pathway distinctions between HL‐AML and NHL‐AML patients. In addition, we analyzed the genetic and functional differences between HL‐AML and NHL‐AML by analyzing the OHSU database. Integrating proteomics and transcriptomics, we identified a significant enrichment of the aberrant NF‐kappa B pathway in HL‐AML. This prompted us to validate whether NF‐kappa B inhibition could represent an effective therapeutic strategy for HL‐AML. So far, the most successful method to inhibit the NF‐kB pathway is to block proteasomal degradation, thereby preventing NF‐kB activation.
Bortezomib is a proteasome inhibitor with broad anti‐tumor activity, exerting its effects by activating multiple signaling cascades, most notably the NF‐kappa B pathway, to induce apoptosis [ 21 ]. Clinically, bortezomib is primarily used in the treatment of multiple myeloma and lymphoma [ 22 , 23 ]. In addition to its role as a proteasome inhibitor, bortezomib also functions as an NF‐kappa B inhibitor. While it demonstrates only modest efficacy as a monotherapy for AML, it is frequently employed in combination with other chemotherapeutic agents for enhanced therapeutic outcomes in leukemia [ 24 , 25 ]. For instance, bortezomib and belinostat have been shown to act synergistically by suppressing NF‐kappa B activity in leukemia cells [ 26 ]. Furthermore, in vivo administration of bortezomib has been demonstrated to inhibit NF‐kappa B activity in leukemia cells [ 27 ]. However, scant research has evaluated the therapeutic effects of bortezomib in HL‐AML. Although monotherapy Phase I studies with bortezomib in relapsed/refractory leukemia children indicate limited hematologic improvement, collaborative usage of bortezomib with other compounds demonstrates potent anti‐leukemic activity against AML and suggests potential clinical utility [ 13 , 27 ]. In addition, the combination of bortezomib, idarubicin, and cytarabine improves overall survival in AML with a favorable safety profile [ 28 ].
Hence, further investigation into the combined use of bortezomib and other compounds for AML therapy is warranted. Cytarabine in combination with anthracycline composition regimen is the standard of care for primary AML [ 29 , 30 , 31 ]. We applied bortezomib in combination with DA regimen in a PDX model and surprisingly found that this regimen had a significant killing effect. Additionally, in clinical practice, we administered a combination regimen of bortezomib with DA regimen to two patients diagnosed with HL‐AML. The first patient experienced relapse after five cycles of standard chemotherapy but achieved disease remission following the combination therapy. The second patient, who had previously received multiple chemotherapy regimens without achieving complete remission, attained complete remission after one cycle of the bortezomib–DA combination therapy. Although only two patients have received this regimen so far and further clinical data are required to confirm its efficacy, the remarkable clinical responses observed in these cases provide compelling evidence for conducting more rigorous clinical trials. These trials aim to evaluate bortezomib in combination with the DA regimen as a potential therapeutic strategy for HL‐AML.
Overall, integrated proteomics and transcriptomics revealed the aberrant protein expression profiles and gene expression profiles of HL‐AML and further revealed a significant enrichment of the NF‐kappa B pathway in HL‐AML, which was suggested as a new therapeutic target. The killing effect of bortezomib on HL‐AML cells was verified in vitro and tested in a PDX model with bortezomib in combination with DA regimen (Figure 5B ). Our investigation advocates for a viable combinatorial therapeutic approach, bortezomib in combination with DA regimen combination therapeutic strategy, to improve the treatment of HL‐AML.
Conclusion
In summary, the combination of proteomic and transcriptomic analyses unveiled aberrant protein and gene expression profiles in HL‐AML. Notably, a significant enrichment of the NF‐kappa B pathway in HL‐AML emerged, indicating a novel therapeutic target. Our study also proposes a viable combination therapeutic strategy involving bortezomib in conjunction with the DA regimen to enhance the treatment of HL‐AML.
Author Contributions
Jinxian Wu: formal analysis (equal), project administration (equal), writing – original draft (equal). Xinqi Li: project administration (equal), writing – original draft (equal). Bei Xiong: project administration (equal), writing – original draft (equal). Wanyue Yin: resources (equal), supervision (equal). Ruihang Li: resources (equal), supervision (equal). Guopeng Chen: data curation (equal), visualization (equal). Linlu Ma: data curation (equal), visualization (equal). Xiqin Tong: data curation (equal), visualization (equal). Xiaoyan Liu: conceptualization (equal), funding acquisition (equal). Fuling Zhou: conceptualization (equal), funding acquisition (equal).
Ethics Statement
The study was approved by the Ethics Committee of Zhongnan Hospital of Wuhan University, and written informed consent forms were obtained from all participants prior to bone marrow puncture.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Data S1.
Acknowledgments
This study was supported by the Natural Science Foundation of China (NSFC) [grant number82370176,82470117,81900116],the Translational Medicine and Cross‐disciplinary Fund of Zhongnan Hospital [grant number ZNJZ202215], and the Hubei Provincial Key Research and Development Program [grant number CZKYXM2023036JZ].
Data Availability Statement
The datasets of this study could be obtained from the corresponding author upon reasonable request and found in the publicly OHSU‐AML project (Oregon Health and Science University, accessed through https://www.cbioportal.org/ ).
References
1 H. Dohner , D. J. Weisdorf , and C. D. Bloomfield , “ Acute Myeloid Leukemia ,” New England Journal of Medicine 373 ( 2015 ): 1136 – 1152 . 26376137 10.1056/NEJMra1406184
2 E. Estey and H. Dohner , “ Acute myeloid leukaemia ,” Lancet 368 ( 2006 ): 1894 – 1907 . 17126723 10.1016/S0140-6736(06)69780-8
3 S. Bertoli , M. Picard , E. Berard , et al., “ Dexamethasone in Hyperleukocytic Acute Myeloid Leukemia ,” Haematologica 103 ( 2018 ): 988 – 998 . 29519869 10.3324/haematol.2017.184267 PMC6058767
4 C. Rollig and G. Ehninger , “ How I Treat Hyperleukocytosis in Acute Myeloid Leukemia ,” Blood 125 ( 2015 ): 3246 – 3252 . 25778528 10.1182/blood-2014-10-551507
5 A. M. Ali , A. E. Mirrakhimov , C. N. Abboud , and A. F. Cashen , “ Leukostasis in Adult Acute Hyperleukocytic Leukemia: A clinician's Digest ,” Hematological Oncology 34 ( 2016 ): 69 – 78 . 27018197 10.1002/hon.2292
6 R. M. Shallis , M. Stahl , J. P. Bewersdorf , J. E. Hendrickson , and A. M. Zeidan , “ Leukocytapheresis for Patients With Acute Myeloid Leukemia Presenting With Hyperleukocytosis and Leukostasis: A Contemporary Appraisal of Outcomes and Benefits ,” Expert Review of Hematology 13 ( 2020 ): 489 – 499 . 32248712 10.1080/17474086.2020.1751609
7 C. Ganzel , J. Becker , P. D. Mintz , H. M. Lazarus , and J. M. Rowe , “ Hyperleukocytosis, Leukostasis and Leukapheresis: Practice Management ,” Blood Reviews 26 ( 2012 ): 117 – 122 . 22364832 10.1016/j.blre.2012.01.003
8 P. Porcu , L. D. Cripe , E. W. Ng , et al., “ Hyperleukocytic Leukemias and Leukostasis: A Review of Pathophysiology, Clinical Presentation and Management ,” Leukemia & Lymphoma 39 ( 2000 ): 1 – 18 . 10975379 10.3109/10428190009053534
9 S. Kittivisuit , N. Jongthitinon , P. Sripornsawan , et al., “ Hyperleukocytosis in Childhood Acute Leukemia: Early Complications and Survival Outcomes ,” Cancers (Basel) 15 ( 2023 ): 15 . 10.3390/cancers15123072 PMC10295972 37370683
10 N. Zhang , X. Liu , J. Wu , et al., “ Serum Proteomics Screening Intercellular Adhesion Molecule‐2 Improves Intermediate‐Risk Stratification in Acute Myeloid Leukemia ,” Therapeutic Advances in Hematology 13 ( 2022 ): 20406207221132346 . 36324489 10.1177/20406207221132346 PMC9619266
11 P. R. Srinivas , M. Verma , Y. Zhao , and S. Srivastava , “ Proteomics for Cancer Biomarker Discovery ,” Clinical Chemistry 48 ( 2002 ): 1160 – 1169 . 12142368
12 H. Gu , J. M. Ren , X. Jia , et al., “ Quantitative Profiling of Post‐Translational Modifications by Immunoaffinity Enrichment and LC‐MS/MS in Cancer Serum Without Immunodepletion ,” Molecular & Cellular Proteomics 15 ( 2016 ): 692 – 702 . 26635363 10.1074/mcp.O115.052266 PMC4739682
13 C. M. Csizmar , D. H. Kim , and Z. Sachs , “ The Role of the Proteasome in AML ,” Blood Cancer Journal 6 ( 2016 ): e503 . 27911437 10.1038/bcj.2016.112 PMC5223148
14 M. Cavo , “ Proteasome Inhibitor Bortezomib for the Treatment of Multiple Myeloma ,” Leukemia 20 ( 2006 ): 1341 – 1352 . 16810203 10.1038/sj.leu.2404278
15 H. Dohner , E. Estey , D. Grimwade , et al., “ Diagnosis and Management of AML in Adults: 2017 ELN Recommendations From an International Expert Panel ,” Blood 129 ( 2017 ): 424 – 447 . 27895058 10.1182/blood-2016-08-733196 PMC5291965
16 H. Dohner , E. H. Estey , S. Amadori , et al., “ Diagnosis and Management of Acute Myeloid Leukemia in Adults: Recommendations From an International Expert Panel, on Behalf of the European LeukemiaNet ,” Blood 115 ( 2010 ): 453 – 474 . 19880497 10.1182/blood-2009-07-235358
17 H. Dombret and C. Gardin , “ An Update of Current Treatments for Adult Acute Myeloid Leukemia ,” Blood 127 ( 2016 ): 53 – 61 . 26660429 10.1182/blood-2015-08-604520 PMC4705610
18 M. Stahl , R. M. Shallis , W. Wei , et al., “ Management of Hyperleukocytosis and Impact of Leukapheresis Among Patients With Acute Myeloid Leukemia (AML) on Short‐ and Long‐Term Clinical Outcomes: A Large, Retrospective, Multicenter, International Study ,” Leukemia 34 ( 2020 ): 3149 – 3160 . 32132655 10.1038/s41375-020-0783-3 PMC8155811
19 S. Oberoi , T. Lehrnbecher , B. Phillips , et al., “ Leukapheresis and Low‐Dose Chemotherapy Do Not Reduce Early Mortality in Acute Myeloid Leukemia Hyperleukocytosis: A Systematic Review and Meta‐Analysis ,” Leukemia Research 38 ( 2014 ): 460 – 468 . 24472688 10.1016/j.leukres.2014.01.004
20 F. Pastore , A. Pastore , G. Wittmann , W. Hiddemann , and K. Spiekermann , “ The Role of Therapeutic Leukapheresis in Hyperleukocytotic AML ,” PLoS One 9 ( 2014 ): e95062 . 24733550 10.1371/journal.pone.0095062 PMC3986260
21 E. P. Jane , D. R. Premkumar , and I. F. Pollack , “ Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF‐kappaB Signaling Pathway ,” Molecular Cancer Therapeutics 10 ( 2011 ): 198 – 208 . 21220502 10.1158/1535-7163.MCT-10-0725 PMC3075591
22 R. C. Kane , R. Dagher , A. Farrell , et al., “ Bortezomib for the Treatment of Mantle Cell Lymphoma ,” Clinical Cancer Research 13 ( 2007 ): 5291 – 5294 . 17875757 10.1158/1078-0432.CCR-07-0871
23 D. Chauhan , T. Hideshima , C. Mitsiades , P. Richardson , and K. C. Anderson , “ Proteasome Inhibitor Therapy in Multiple Myeloma ,” Molecular Cancer Therapeutics 4 ( 2005 ): 686 – 692 . 15827343 10.1158/1535-7163.MCT-04-0338
24 E. C. Attar , J. L. Johnson , P. C. Amrein , et al., “ Bortezomib Added to Daunorubicin and Cytarabine During Induction Therapy and to Intermediate‐Dose Cytarabine for Consolidation in Patients With Previously Untreated Acute Myeloid Leukemia Age 60 to 75 Years: CALGB (Alliance) Study 10502 ,” Journal of Clinical Oncology 31 ( 2013 ): 923 – 929 . 23129738 10.1200/JCO.2012.45.2177 PMC3577952
25 C. Yu , M. Rahmani , D. Conrad , M. Subler , P. Dent , and S. Grant , “ The Proteasome Inhibitor Bortezomib Interacts Synergistically With Histone Deacetylase Inhibitors to Induce Apoptosis in Bcr/Abl+ Cells Sensitive and Resistant to STI571 ,” Blood 102 ( 2003 ): 3765 – 3774 . 12893773 10.1182/blood-2003-03-0737
26 Y. Dai , S. Chen , L. Wang , et al., “ Bortezomib Interacts Synergistically With Belinostat in Human Acute Myeloid Leukaemia and Acute Lymphoblastic Leukaemia Cells in Association With Perturbations in NF‐kappaB and Bim ,” British Journal of Haematology 153 ( 2011 ): 222 – 235 . 21375523 10.1111/j.1365-2141.2011.08591.x PMC3358826
27 T. M. Horton , D. Pati , S. E. Plon , et al., “ A Phase 1 Study of the Proteasome Inhibitor Bortezomib in Pediatric Patients With Refractory Leukemia: A Children's Oncology Group Study ,” Clinical Cancer Research 13 ( 2007 ): 1516 – 1522 . 17332297 10.1158/1078-0432.CCR-06-2173
28 E. C. Attar , D. J. De Angelo , J. G. Supko , et al., “ Phase I and Pharmacokinetic Study of Bortezomib in Combination With Idarubicin and Cytarabine in Patients With Acute Myelogenous Leukemia ,” Clinical Cancer Research 14 ( 2008 ): 1446 – 1454 . 18316568 10.1158/1078-0432.CCR-07-4626
29 H. Liu , “ Emerging Agents and Regimens for AML ,” Journal of Hematology & Oncology 14 ( 2021 ): 49 . 33757574 10.1186/s13045-021-01062-w PMC7989091
30 A. Burnett , M. Wetzler , and B. Lowenberg , “ Therapeutic Advances in Acute Myeloid Leukemia ,” Journal of Clinical Oncology 29 ( 2011 ): 487 – 494 . 21220605 10.1200/JCO.2010.30.1820
31 T. Robak and A. Wierzbowska , “ Current and Emerging Therapies for Acute Myeloid Leukemia ,” Clinical Therapeutics 31 , no. Pt 2 ( 2009 ): 2349 – 2370 . 20110045 10.1016/j.clinthera.2009.11.017