RARγ activation sensitizes human myeloma cells to carfilzomib treatment through the OAS-RNase L innate immune pathway.

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) are highly efficacious for patients with multiple myeloma (MM). However, relapses are frequent, and acquired resistance to PI treatment emerges in most patients. Here, we performed a high-throughput screen of 1855 Food and Drug Administration (FDA)-approved drugs and identified all-trans retinoic acid (ATRA), which alone has no antimyeloma effect, as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and resensitized Cfz-resistant MM cells to Cfz in vitro. ATRA activated retinoic acid receptor (RAR)γ and interferon-ß response pathway, leading to upregulated expression of IRF1. IRF1 in turn initiated the transcription of OAS1, which synthesized 2-5A upon binding to double-stranded RNA (dsRNA) induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death. Similar to ATRA, BMS961, a selective RARγ agonist, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo. In support of these findings, analyses of large datasets of patients' gene profiling showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment. Thus, this study highlights the potential for RARγ agonists to sensitize and overcome MM resistance to Cfz treatment in patients.

Aptamer-armed nanostructures improve the chemotherapy outcome of triple-negative breast cancer.

Triple-negative breast cancer is an aggressive subtype of breast cancer that is primarily treated using systemic chemotherapy due to the lack of a specific cell surface marker for drug delivery. Cancer cell-specific aptamer-mediated drug delivery is a promising targeted chemotherapy for marker-unknown cancers. Using a poorly differentiated carcinoma cell-specific DNA aptamer (PDGC21T), we formed a self-assembling circinate DNA nanoparticle (Apt21TNP) that binds triple-negative breast cancer cells. Using our previously designed pH-sensitive dendrimer-conjugated doxorubicin (DDOX) as the payload, we found that each nanoparticle loaded 30 doxorubicin molecules to form an Apt21TNP-DDOX nanomedicine that is stable in human plasma. Upon cell binding, Apt21TNP-DDOX is internalized by triple-negative breast cancer cells through the macropinocytosis pathway. Once inside cells, the low pH microenvironment in lysosomes induces doxorubicin drug payload release from Apt21TNP-DDOX. Our in vitro studies demonstrate that Apt21TNP-DDOX can preferentially bind triple-negative breast cancer cells to induce cell death. Furthermore, we show that Apt21TNP-DDOX can accumulate in subcutaneous MDA-MB-231 tumors in mice following systemic administration to reduce tumor burden, minimize side effects, and improve animal survival. Together, our results demonstrate that Apt21TNP-mediated doxorubicin delivery is a potent, targeted chemotherapy for triple-negative breast cancer that may alleviate side effects in patients.

MIF as a biomarker and therapeutic target for overcoming resistance to proteasome inhibitors in human myeloma.

Multiple myeloma (MM) remains largely incurable despite significant advances in biotherapy and chemotherapy. The development of drug resistance is a major problem in MM management. Macrophage migration inhibitory factor (MIF) expression was significantly higher in purified MM cells from relapsed patients than those with sustained response, and MM patients with high MIF had significantly shorter progression-free survival (PFS) and overall survival (OS). MM cell lines also express high levels of MIF, and knocking out MIF made them more sensitive to proteasome inhibitor (PI)-induced apoptosis not observed with other chemotherapy drugs. Mechanistic studies showed that MIF protects MM cells from PI-induced apoptosis by maintaining mitochondrial function via suppression of superoxide production in response to PIs. Specifically, MIF, in the form of a homotrimer, acts as a chaperone for superoxide dismutase 1 (SOD1) to suppress PI-induced SOD1 misfolding and to maintain SOD1 activity. MIF inhibitor 4-iodo-6-phenylpyrimidine and homotrimer disrupter ebselen, which do not kill MM cells, enhanced PI-induced SOD1 misfolding and loss of function, resulting in significantly more cell death in both cell lines and primary MM cells. More importantly, inhibiting MIF activity in vivo displayed synergistic antitumor activity with PIs and resensitized PI-resistant MM cells to treatment. In support of these findings, gene-profiling data showed a significantly negative correlation between MIF and SOD1 expression and response to PI treatment in patients with MM. This study shows that MIF plays a crucial role in MM sensitivity to PIs and suggests that targeting MIF may be a promising strategy to (re)sensitize MM to the treatment.

EpCAM-Targeting Aptamer Radiotracer for Tumor-Specific PET Imaging.

Noninvasive in vivo imaging to measure the expression of EpCAM, a biomarker overexpressed in the majority of carcinoma tumors and metastatic lesions, is highly desirable for accurate tumor staging and therapy evaluation. Here, we report the use of an aptamer radiotracer to enable tumor-specific EpCAM-targeting PET imaging. Oligonucleotide aptamers are small molecular ligands that specifically bind with high affinity to their target molecules. For specific tumor imaging, an aptamer radiotracer was formulated by chelating a 64Cu isotope and DOTA-PEGylated aptamer sequence to target EpCAM. In vitro cell uptake assays demonstrated that the aptamer radiotracer specifically bound EpCAM-expressing breast cancer cells but did not react with off-target tumor cells. For in vivo tumor imaging, aptamer radiotracer was systemically administered into xenograft mice. MicroPET/CT scans revealed that the aptamer radiotracer rapidly highlighted xenograft tumors derived from MDA-MB-231 breast cancer cells (EpCAM positive) as early as 2 h postadministration with a gradually increasing tumor uptake signal that peaked at 24 h but not in lymphoma 937 tumors (EpCAM negative). In contrast, nonspecific background signals in the liver and kidneys were rapidly decreased postadministration. This proof-of-concept study demonstrates the utility of aptamer radiotracers for tumor-specific PET imaging.

G6PD deficiency and severity of COVID19 pneumonia and acute respiratory distress syndrome: tip of the iceberg?

The severe pneumonia caused by the human coronavirus (hCoV)-SARS-CoV-2 has inflicted heavy casualties, especially among the elderly and those with co-morbid illnesses irrespective of their age. The high mortality in African-Americans and males, in general, raises the concern for a possible X-linked mediated process that could affect the viral pathogenesis and the immune system. We hypothesized that G6PD, the most common X-linked enzyme deficiency, associated with redox status, may have a role in severity of pneumonia. Retrospective chart review was performed in hospitalized patients with COVID19 pneumonia needing supplemental oxygen. A total of 17 patients were evaluated: six with G6PD deficiency (G6PDd) and 11 with normal levels. The two groups (normal and G6PDd) were comparable in terms of age, sex, co-morbidities, and laboratory parameters-LDH, IL-6, CRP, and ferritin, respectively. Thirteen patients needed ventilatory support ; 8 in the normal group and 5 in the G6PDd group (72% vs.83%). The main differences indicating increasing severity in normal vs. G6PDd groups included G6PD levels (12.2 vs. 5.6, P = 0.0002), PaO2/FiO2 ratio (159 vs. 108, P = 0.05), days on mechanical ventilation (10.25 vs. 21 days P = 0.04), hemoglobin level (10 vs. 8.1 P = 0.03), and hematocrit (32 vs. 26 P = 0.015). Only one patient with G6PDd died; 16 were discharged home. Our clinical series ascribes a possible biological role for G6PDd in SARS-CoV2 viral proliferation. It is imperative that further studies are performed to understand the interplay between the viral and host factors in G6PDd that may lead to disparity in outcomes. KEY POINTS: • COVID19 studies show higher mortality in men, due to severe pneumonia and ARDS, indicating possible X-linked mediated differences • G6PD, the most common X-linked enzymopathy, highly prevalent in African Americans and Italians, maintains redox homeostasis. • Preclinical studies using G6PD deficient (G6PDd) cells infected with human coronavirus (hCoV), show impaired cellular responses, viral proliferation and worsening oxidative damage. • Retrospective chart review in hospitalized patients with COVID19 pneumonia needing supplemental oxygen shows differences between the two groups (Normal and G6PDd) in hematological indices; the G6PDdgroup demonstrated prolonged PaO2/FiO2 ratio, and longer days on mechanical ventilation indicating the severity of the pneumonia.

Neutralizing Aptamers Block S/RBD-ACE2 Interactions and Prevent Host Cell Infection.

The receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 spike (S) protein plays a central role in mediating the first step of virus infection to cause disease: virus binding to angiotensin-converting enzyme 2 (ACE2) receptors on human host cells. Therefore, S/RBD is an ideal target for blocking and neutralization therapies to prevent and treat coronavirus disease 2019 (COVID-19). Using a target-based selection approach, we developed oligonucleotide aptamers containing a conserved sequence motif that specifically targets S/RBD. Synthetic aptamers had high binding affinity for S/RBD-coated virus mimics (KD ≈7 nM) and also blocked interaction of S/RBD with ACE2 receptors (IC50 ≈5 nM). Importantly, aptamers were able to neutralize S protein-expressing viral particles and prevent host cell infection, suggesting a promising COVID-19 therapy strategy.

High levels of immunoglobulin expression predict shorter overall survival in patients with acute myeloid leukemia.

OBJECTIVES: It has been believed that immunoglobulins can only be produced by B lymphocytes and plasma cells. We have previously reported that IgG can be expressed in myeloblasts from patients with acute myeloid leukemia (AML) and plays a role in the proliferation and apoptosis of leukemic cells. However, its clinical impact has not been assessed. METHODS: We assessed the expression of different classes of immunoglobulin in peripheral blood and bone marrow samples from 132 AML patients and correlated the levels of expression with clinicopathologic and molecular genetic features, as well as clinical outcome. RESULTS: We found that, in addition to IgG, all classes of immunoglobulin are expressed in myeloblasts, including IgG, IgM, IgA, IgD, IgE, Igκ, and Igλ. The levels of IgG expression (coupled with Igκ or Igλ) are higher than those of IgM, IgA, IgD, and IgE. Using receiver operating characteristic (ROC) curve analysis, we identified two distinct groups of AML patients with differential expression of immunoglobulin and different clinical outcomes. CONCLUSIONS: High levels of immunoglobulin expression are associated with monocytic differentiation, multilineage dysplasia, TET2 and KRAS mutations, and poor overall survival. Assessment of immunoglobulin may serve as a useful marker for prognostic stratification and target therapy.

Aptamer Cocktail to Detect Multiple Species of Mycoplasma in Cell Culture.

Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.

Aptamer-Engineered Natural Killer Cells for Cell-Specific Adaptive Immunotherapy.

Natural killer (NK) cells are a key component of the innate immune system as they can attack cancer cells without prior sensitization. However, due to lack of cell-specific receptors, NK cells are not innately able to perform targeted cancer immunotherapy. Aptamers are short single-stranded oligonucleotides that specifically recognize their targets with high affinity in a similar manner to antibodies. To render NK cells with target-specificity, synthetic CD30-specific aptamers are anchored on cell surfaces to produce aptamer-engineered NK cells (ApEn-NK) without genetic alteration or cell damage. Under surface-anchored aptamer guidance, ApEn-NK specifically bind to CD30-expressing lymphoma cells but do not react to off-target cells. The resulting specific cell binding of ApEn-NK triggers higher apoptosis/death rates of lymphoma cells compared to parental NK cells. Additionally, experiments with primary human NK cells demonstrate the potential of ApEn-NK to specifically target and kill lymphoma cells, thus presenting a potential new approach for targeted immunotherapy by NK cells.

High-Throughput Isolation of Cell Protrusions with Single-Cell Precision for Profiling Subcellular Gene Expression.

Invading cancer cells extend cell protrusions, which guide cancer-cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single-cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high-throughput isolation of cell protrusions at single-cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell-protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single-cell precision for subsequent RNA-Seq analysis, which was further validated by RT-qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.

Self-Assembled Aptamer-Nanomedicine for Targeted Chemotherapy and Gene Therapy.

Chemotherapy is the mainstream treatment of anaplastic large cell lymphoma (ALCL). However, chemotherapy can cause severe adverse effects in patients because it is not ALCL-specific. In this study, a multifunctional aptamer-nanomedicine (Apt-NMed) achieving targeted chemotherapy and gene therapy of ALCL is developed. Apt-NMed is formulated by self-assembly of synthetic oligonucleotides containing CD30-specific aptamer and anaplastic lymphoma kinase (ALK)-specific siRNA followed by self-loading of the chemotherapeutic drug doxorubicin (DOX). Apt-NMed exhibits a well-defined nanostructure (diameter 59 mm) and stability in human serum. Under aptamer guidance, Apt-NMed specifically binds and internalizes targeted ALCL cells. Intracellular delivery of Apt-NMed triggers rapid DOX release for targeted ALCL chemotherapy and intracellular delivery of the ALK-specific siRNA induced ALK oncogene silencing, resulting in combined therapeutic effects. Animal model studies reveal that upon systemic administration, Apt-NMed specifically targets and selectively accumulates in ALCL tumor site, but does not react with off-target tumors in the same xenograft mouse. Importantly, Apt-NMed not only induces significantly higher inhibition in ALCL tumor growth, but also causes fewer or no side effects in treated mice compared to free DOX. Moreover, Apt-NMed treatment markedly improves the survival rate of treated mice, opening a new avenue for precision treatment of ALCL.

Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma.

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37-) in ∼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37-, including TP53 mutation, NF-κBhigh, Mychigh, phosphorylated STAT3high, survivinhigh, p63-, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37- status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37- DLBCL, ICOSLG upregulation in CD37+ GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.

DNA Thioaptamer with Homing Specificity to Lymphoma Bone Marrow Involvement.

Selective drug accumulation in the malignant tissue is a prerequisite for effective cancer treatment. However, most drug molecules and their formulated particles are blocked en route to the destiny tissue due to the existence of multiple biological and physical barriers including the tumor microvessel endothelium. Since the endothelial cells on the surface of the microvessel wall can be modulated by inflammatory cytokines and chemokines secreted by the tumor or stromal cells, an effective drug delivery approach is to enhance interaction between the drug particles and the unique spectrum of surface proteins on the tumor endothelium. In this study, we performed in vivo screening for thioaptamers that bind to the bone marrow endothelium with specificity in a murine model of lymphoma with bone marrow involvement (BMI). The R1 thioaptamer was isolated based on its high homing potency to bones with BMI, and 40-60% less efficiency in accumulation to healthy bones. In cell culture, R1 binds to human umbilical vein endothelial cells (HUVEC) with a high affinity ( Kd ≈ 3 nM), and the binding affinity can be further enhanced when cells were treated with a mixture of lymphoma cell and bone marrow cell conditioned media. Cellular uptake of R1 is through clathrin-mediated endocytosis. Conjugating R1 on to the surface of liposomal doxorubicin nanoparticles resulted in 2-3-fold increase in drug accumulation in lymphoma BMI. Taking together, we have successfully identified a thioaptamer that preferentially binds to the endothelium of lymphoma BMI. It can serve as an affinity moiety for targeted delivery of drug particles to the disease organ.

Ultrasensitive Magnetic Nanoparticle Detector for Biosensor Applications.

Ta/Ru/Co/Ru/Co/Cu/Co/Ni80Fe20/Ta spin-valve giant magnetoresistive (GMR) multilayers were deposited using UHV magnetron sputtering and optimized to achieve a 13% GMR ratio before patterning. The GMR multilayer was patterned into 12 sensor arrays using a combination of e-beam and optical lithographies. Arrays were constructed with 400 nm × 400 nm and 400 nm × 200 nm sensors for the detection of reporter nanoparticles. Nanoparticle detection was based on measuring the shift in high-to-low resistance switching field of the GMR sensors in the presence of magnetic particle(s). Due to shape anisotropy and the corresponding demag field, the resistance state switching fields were significantly larger and the switching field distribution significantly broader in the 400 nm × 200 nm sensors as compared to the 400 nm × 400 nm sensors. Thus, sensor arrays with 400 nm × 400 nm dimensions were used for the demonstration of particle detection. Detection of a single 225 nm Fe3O4 magnetic nanoparticle and a small number (~10) of 100 nm nanoparticles was demonstrated. With appropriate functionalization for biomolecular recognition, submicron GMR sensor arrays can serve as the basis of ultrasensitive chemical and biological sensors.

Aptamers: versatile molecular recognition probes for cancer detection.

In the past two decades, aptamers have emerged as a novel class of molecular recognition probes comprising uniquely-folded short RNA or single-stranded DNA oligonucleotides that bind to their cognate targets with high specificity and affinity. Aptamers, often referred to as "chemical antibodies", possess several highly desirable features for clinical use. They can be chemically synthesized and are easily conjugated to a wide range of reporters for different applications, and are able to rapidly penetrate tissues. These advantages significantly enhance their clinical applicability, and render them excellent alternatives to antibody-based probes in cancer diagnostics and therapeutics. Aptamer probes based on fluorescence, colorimetry, magnetism, electrochemistry, and in conjunction with nanomaterials (e.g., nanoparticles, quantum dots, single-walled carbon nanotubes, and magnetic nanoparticles) have provided novel ultrasensitive cancer diagnostic strategies and assays. Furthermore, promising aptamer targeted-multimodal tumor imaging probes have been recently developed in conjunction with fluorescence, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). The capabilities of the aptamer-based platforms described herein underscore the great potential they hold for the future of cancer detection. In this review, we highlight the most prominent recent developments in this rapidly advancing field.

Dynamic balance of multiple myeloma clonogenic side population cell percentages controlled by environmental conditions.

Cancer stem cells are key drivers of tumor progression and disease recurrence in multiple myeloma (MM). However, little is known about the regulation of MM stem cells. Here, we show that a population of MM cells, known as the side population (SP), exhibits stem-like properties. Cells that constitute the SP in primary MM isolates are negative or seldom expressed for CD138 and CD20 markers. In addition, the SP population contains stem cells that belong to the same lineage as the mature neoplastic plasma cells. Importantly, our data indicate that the SP and nonside population (NSP) percentages in heterogeneous MM cells are balanced, and that this balance can be achieved through a prolonged in vitro culture. Furthermore, we show that SP cells, with confirmed molecular characteristics of MM stem cells, can be regenerated from purified NSP cell populations. We also show that the percentage of SP cells can be enhanced by the hypoxic stress, which is frequently observed within MM tumors. Finally, hypoxic stress enhanced the expression of transforming growth factor ß1 (TGF-ß1) and blocking the TGF-ß1 signaling pathway inhibited the NSP dedifferentiation. Taken together, these findings indicate that the balance between MM SP and NSP is regulated by environmental factors and TGF-ß1 pathway is involved in hypoxia-induced increase of SP population. Understanding the mechanisms that facilitate SP maintenance will accelerate the design of novel therapeutics aimed at controlling these cells in MM.

SDF-1α stiffens myeloma bone marrow mesenchymal stromal cells through the activation of RhoA-ROCK-Myosin II.

Multiple myeloma (MM) is a B lymphocyte malignancy that remains incurable despite extensive research efforts. This is due, in part, to frequent disease recurrences associated with the persistence of myeloma cancer stem cells (mCSCs). Bone marrow mesenchymal stromal cells (BMSCs) play critical roles in supporting mCSCs through genetic or biochemical alterations. Previously, we identified mechanical distinctions between BMSCs isolated from MM patients (mBMSCs) and those present in the BM of healthy individuals (nBMSCs). These properties of mBMSC contributed to their ability to preferentially support mCSCs. To further illustrate mechanisms underlying the differences between mBMSCs and nBMSCs, here we report that (i) mBMSCs express an abnormal, constitutively high level of phosphorylated Myosin II, which leads to stiffer membrane mechanics, (ii) mBMSCs are more sensitive to SDF-1α-induced activation of MYL2 through the G(i./o)-PI3K-RhoA-ROCK-Myosin II signaling pathway, affecting Young's modulus in BMSCs and (iii) activated Myosin II confers increased cell contractile potential, leading to enhanced collagen matrix remodeling and promoting the cell-cell interaction between mCSCs and mBMSCs. Together, our findings suggest that interfering with SDF-1α signaling may serve as a new therapeutic approach for eliminating mCSCs by disrupting their interaction with mBMSCs.

Aptamers and their applications in nanomedicine.

Aptamers are composed of short RNA or single-stranded DNA sequences that, when folded into their unique 3D conformation, can bind to their targets with high specificity and affinity. Although functionally similar to protein antibodies, oligonucleotide aptamers offer several advantages over protein antibodies in biomedical and clinical applications. Through the enhanced permeability and retention effect, nanomedicines can improve the therapeutic index of a treatment and reduce side effects by enhancing accumulation at the disease site. However, this targets tumors passively and, thus, may not be ideal for targeted therapy. To construct ligand-directed "active targeting" nanobased delivery systems, aptamer-equipped nanomedicines have been tested for in vitro diagnosis, in vivo imaging, targeted cancer therapy, theranostic approaches, sub-cellular molecule detection, food safety, and environmental monitoring. This review focuses on the development of aptamer-conjugated nanomedicines and their application for in vivo imaging, targeted therapy, and theranostics.

Clinical features, tumor biology, and prognosis associated with MYC rearrangement and Myc overexpression in diffuse large B-cell lymphoma patients treated with rituximab-CHOP.

MYC dysregulation, including MYC gene rearrangement and Myc protein overexpression, is of increasing clinical importance in diffuse large B-cell lymphoma (DLBCL). However, the roles of MYC and the relative importance of rearrangement vs overexpression remain to be refined. Gaining knowledge about the tumor biology associated with MYC dysregulation is important to understand the roles of MYC and MYC-associated biology in lymphomagenesis. In this study, we determined MYC rearrangement status (n=344) and Myc expression (n=535) in a well-characterized DLBCL cohort, individually assessed the clinical and pathobiological features of patients with MYC rearrangement and Myc protein overexpression, and analyzed the prognosis and gene expression profiling signatures associated with these MYC abnormalities in germinal center B-cell-like and activated B-cell-like DLBCL. Our results showed that the prognostic importance of MYC rearrangement vs Myc overexpression is significantly different in germinal center B-cell-like vs activated B-cell-like DLBCL. In germinal center B-cell-like DLBCL, MYC-rearranged germinal center B-cell-like DLBCL patients with Myc overexpression significantly contributed to the clinical, biological, and prognostic characteristics of the overall Myc-overexpressing germinal center B-cell-like DLBCL group. In contrast, in activated B-cell-like DLBCL, the occurrence, clinical and biological features, and prognosis of Myc overexpression were independent of MYC rearrangement. High Myc levels and Myc-independent mechanisms, either tumor cell intrinsic or related to tumor microenvironment, conferred significantly worse survival to MYC-rearranged germinal center B-cell-like DLBCL patients, even among Myc(high)Bcl-2(high) DLBCL patients. This study provides new insight into the tumor biology and prognostic effects associated with MYC dysregulation and suggest that detection of both MYC translocations and evaluation of Myc and Bcl-2 expression is necessary to predict the prognosis of DLBCL patients.