IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHC CII complexes.

In diffuse large B-cell lymphoma (DLBCL), the transcription factor IRF8 is the target of a series of potentially oncogenic events, including, chromosomal translocation, focal amplification, and super-enhancer perturbations. IRF8 is also frequently mutant in DLBCL, but how these variants contribute to lymphomagenesis is unknown. We modeled IRF8 mutations in DLBCL and found that they did not meaningfully impact cell fitness. Instead, IRF8 mutants, mapping either to the DNA-binding domain (DBD) or c-terminal tail, displayed diminished transcription activity towards CIITA, a direct IRF8 target. In primary DLBCL, IRF8 mutations were mutually exclusive with mutations in genes involved in antigen presentation. Concordantly, expression of IRF8 mutants in murine B cell lymphomas uniformly suppressed CD4, but not CD8, activation elicited by antigen presentation. Unexpectedly, IRF8 mutation did not modify MHC CII expression on the cell surface, rather it downmodulated CD74 and HLA- DM, intracellular regulators of antigen peptide processing/loading in the MHC CII complex. These changes were functionally relevant as, in comparison to IRF8 WT, mice harboring IRF8 mutant lymphomas displayed a significantly higher tumor burden, in association with a substantial remodeling of the tumor microenvironment (TME), typified by depletion of CD4, CD8, Th1 and NK cells, and increase in T-regs and Tfh cells. Importantly, the clinical and immune phenotypes of IRF8-mutant lymphomas were rescued in vivo by ectopic expression of CD74. Deconvolution of bulk RNAseq data from primary human DLBCL recapitulated part of the immune remodeling detected in mice and pointed to depletion of dendritic cells as another feature of IRF8 mutant TME. We concluded that IRF8 mutations contribute to DLBCL biology by facilitating immune escape.

The Emerging Role of Extracellular Vesicle-Associated RNAs in the Multiple Myeloma Microenvironment.

Multiple myeloma (MM) is a cancer of terminally differentiated plasma cells (PCs) that develop at multiple sites within the bone marrow (BM). MM is treatable but rarely curable because of the frequent emergence of drug resistance and relapse. Increasing evidence indicates that the BM microenvironment plays a major role in supporting MM-PC survival and resistance to therapy. The BM microenvironment is a complex milieu containing hematopoietic cells, stromal cells, endothelial cells, immune cells, osteoclasts and osteoblasts, all contributing to the pathobiology of MM, including PC proliferation, escape from immune surveillance, angiogenesis and bone disease development. Small extracellular vesicles (EVs) are heterogenous lipid structures released by all cell types and mediate local and distal cellular communication. In MM, EVs are key mediators of the cross-talk between PCs and the surrounding microenvironment because of their ability to deliver bioactive cargo molecules such as lipids, mRNAs, non-coding regulatory RNA and proteins. Hence, MM-EVs highly contribute to establish a tumor-supportive BM niche that impacts MM pathogenesis and disease progression. In this review, we will first highlight the effects of RNA-containing, MM-derived EVs on the several cellular compartments within the BM microenvironment that play a role in the different aspects of MM pathology. We will also touch on the prospective use of MM-EV-associated non-coding RNAs as clinical biomarkers in the context of "liquid biopsy" in light of their importance as a promising tool in MM diagnosis, prognosis and prediction of drug resistance.

Daratumumab induces mechanisms of immune activation through CD38+ NK cell targeting.

Daratumumab (Dara), a multiple myeloma (MM) therapy, is an antibody against the surface receptor CD38, which is expressed not only on plasma cells but also on NK cells and monocytes. Correlative data have highlighted the immune-modulatory role of Dara, despite the paradoxical observation that Dara regimens decrease the frequency of total NK cells. Here we show that, despite this reduction, NK cells play a pivotal role in Dara anti-MM activity. CD38 on NK cells is essential for Dara-induced immune modulation, and its expression is restricted to NK cells with effector function. We also show that Dara induces rapid CD38 protein degradation associated with NK cell activation, leaving an activated CD38-negative NK cell population. CD38+ NK cell targeting by Dara also promotes monocyte activation, inducing an increase in T-cell costimulatory molecules (CD86/80) and enhancing anti-MM phagocytosis activity ex vivo and in vivo. In support of Dara's immunomodulating role, we show that MM patients that discontinued Dara therapy because of progression maintain targetable unmutated surface CD38 expression on their MM cells, but retain effector cells with impaired cellular immune function. In summary, we report that CD38+ NK cells may be an unexplored therapeutic target for priming the immune system of MM patients.

Extracellular Vesicles in Hematological Malignancies: From Biomarkers to Therapeutic Tools.

Small extracellular vesicles (EVs) are a heterogenous group of lipid particles released by all cell types in physiological and pathological states. In hematological malignancies, tumor-derived EVs are critical players in mediating intercellular communications through the transfer of genetic materials and proteins between neoplastic cells themselves and to several components of the bone marrow microenvironment, rendering the latter a "stronger" niche supporting cancer cell proliferation, drug resistance, and escape from immune surveillance. In this context, the molecular cargoes of tumor-derived EVs reflect the nature and status of the cells of origin, making them specific therapeutic targets. Another important characteristic of EVs in hematological malignancies is their use as a potential "liquid biopsy" because of their high abundance in biofluids and their ability to protect their molecular cargoes from nuclease and protease degradation. Liquid biopsies are non-invasive blood tests that provide a molecular profiling clinical tool as an alternative method of disease stratification, especially in cancer patients where solid biopsies have limited accessibility. They offer accurate diagnoses and identify specific biomarkers for monitoring of disease progression and response to treatment. In this review, we will focus on the role of EVs in the most prevalent hematological malignancies, particularly on their prospective use as biomarkers in the context of liquid biopsies, as well as their molecular signature that identifies them as specific therapeutic targets for inhibiting cancer progression. We will also highlight their roles in modulating the immune response by acting as both immunosuppressors and activators of anti-tumor immunity.

MiR-16 regulates crosstalk in NF-κB tolerogenic inflammatory signaling between myeloma cells and bone marrow macrophages.

High levels of circulating miR-16 in the serum of multiple myeloma (MM) patients are independently associated with longer survival. Although the tumor suppressor function of intracellular miR-16 in MM plasma cells (PCs) has been elucidated, its extracellular role in maintaining a nonsupportive cancer microenvironment has not been fully explored. Here, we show that miR-16 is abundantly released by MM cells through extracellular vesicles (EVs) and that differences in its intracellular expression as associated with chromosome 13 deletion (Del13) are correlated to extracellular miR-16 levels. We also demonstrate that EVs isolated from MM patients and from the conditioned media of MM-PCs carrying Del13 more strongly differentiate circulating monocytes to M2-tumor supportive macrophages (TAMs), compared with MM-PCs without this chromosomal aberration. Mechanistically, our data show that miR-16 directly targets the IKKα/ß complex of the NF-κB canonical pathway, which is critical not only in supporting MM cell growth, but also in polarizing macrophages toward an M2 phenotype. By using a miR-15a-16-1-KO mouse model, we found that loss of the miR-16 cluster supports polarization to M2 macrophages. Finally, we demonstrate the therapeutic benefit of miR-16 overexpression in potentiating the anti-MM activity by a proteasome inhibitor in the presence of MM-resident bone marrow TAM.

Daratumumab induces CD38 internalization and impairs myeloma cell adhesion.

Daratumumab (Dara), a human immunoglobulin G1 kappa (IgG1κ) monoclonal anti-CD38 antibody, has been approved by the U.S. Food and Drug Administration for the treatment of relapsed multiple myeloma (MM) as a single agent as well as in combination with immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI). Although the scientific rationale behind the use of Dara in combination with IMiDs has been extensively explored, the molecular mechanisms underlying Dara-PI regimens have not yet been investigated. Here, we demonstrate that CD38 on the surface of MM cells is rapidly internalized after Dara treatment; we also show that Dara treatment impairs MM cell adhesion, an effect that can be rescued by using the endocytosis inhibitor Dynasore. Finally, we show that Dara potentiates bortezomib (BTZ) killing of MM cells in vitro and in vivo, independent of its function as an immune activator. In conclusion, our data show that Dara impairs MM cell adhesion, which results in an increased sensitivity of MM to proteasome inhibition.

Copper 64-labeled daratumumab as a PET/CT imaging tracer for multiple myeloma.

As a growing number of patients with multiple myeloma (MM) respond to upfront therapies while eventually relapsing in a time frame that is often unpredictable, attention has increasingly focused on developing novel diagnostic criteria to also account for disease dissemination. Positron emission tomography/computed tomography (PET/CT) is often used as a noninvasive monitoring strategy to assess cancer cell dissemination, but because the uptake of the currently used radiotracer 18fluorodeoxyglucose (18F-FDG) is a function of the metabolic activity of both malignant and nonmalignant cells, the results frequently lack sufficient specificity. Radiolabeled antibodies targeting MM tissue may detect disease irrespective of cell metabolism. Hence, we conjugated the clinically significant CD38-directed human antibody daratumumab (Darzalex [Dara]) to the DOTA chelator and labeled it with the positron-emitting radionuclide copper 64 (64Cu; 64Cu-DOTA-Dara). Here, we show that 64Cu-DOTA-Dara can efficiently bind CD38 on the surface of MM cells and was mainly detected in the bones associated with tumor in a MM murine model. We also show that PET/CT based on 64Cu-DOTA-Dara displays a higher resolution and specificity to detect MM cell dissemination than does 18F-FDG PET/CT and was even more sensitive than were bioluminescence signals. We therefore have supporting evidence for using 64Cu-DOTA-Dara as a novel imaging agent for MM.

Effects of Ascorbic Acid on Tax, NF-κB and MMP-9 in Human T-cell Lymphotropic Virus Type 1 Positive Malignant T-Lymphocytes.

BACKGROUND: HTLV1 is a retrovirus that infects CD4-positive cells and leads to Adult T-cell leukemia by constitutive activation of nuclear factor kappa B. Ascorbic acid (AA) is an essential nutrient that possess anti-proliferative and pro-apoptotic activity against a number of malignant cell lines. This study delineates the effect of AA on Tax protein expression as well as NF-κB and MMP9 activity in two HTLV1-positive leukemia cells (HuT-102 and C91-PL). METHODS: The cytotoxic and antiproliferative effect of AA were studied by LDH release and MTT tests, respectively. The proteins expression level was assessed by western blotting. RT-PCR was used to study mRNAs level. Finally, ELISA/EMSA and Zymography were used to evaluate NF-κB and MMP-9 activities, respectively. RESULTS: Cell lines were treated with non-cytotoxic concentrations of AA for 48h and 96h, which resulted in a significant inhibition of proliferation at a concentration of 50µg/ml at 96h in both cell lines. The same concentration inhibited Tax protein expression as well as the NF-κB nuclearization and DNA binding activity. The inhibitory effect of AA on MMP9 protein expression and activity started at 100µg/ml and 50µg/ml in HuT-102 and C91-PL cells respectively, with no effect at the transcriptional levels of MMP-9 in either one of the two cell lines. CONCLUSION: These results indicated that while AA exerted its anti-proliferative effect on the NF- κB activation pathway by suppressing Tax expression, its effects on MMP9 seemed to be independent of this mechanism and follow a different approach.

Targeting the RAS/MAPK pathway with miR-181a in acute myeloid leukemia.

Deregulation of microRNAs' expression frequently occurs in acute myeloid leukemia (AML). Lower miR-181a expression is associated with worse outcomes, but the exact mechanisms by which miR-181a mediates this effect remain elusive. Aberrant activation of the RAS pathway contributes to myeloid leukemogenesis. Here, we report that miR-181a directly binds to 3'-untranslated regions (UTRs); downregulates KRAS, NRAS and MAPK1; and decreases AML growth. The delivery of miR-181a mimics to target AML cells using transferrin-targeting lipopolyplex nanoparticles (NP) increased mature miR-181a; downregulated KRAS, NRAS and MAPK1; and resulted in decreased phosphorylation of the downstream RAS effectors. NP-mediated upregulation of miR-181a led to reduced proliferation, impaired colony formation and increased sensitivity to chemotherapy. Ectopic expression of KRAS, NRAS and MAPK1 attenuated the anti-leukemic activity of miR-181a mimics, thereby validating the relevance of the deregulated miR-181a-RAS network in AML. Finally, treatment with miR-181a-NP in a murine AML model resulted in longer survival compared to mice treated with scramble-NP control. These data support that targeting the RAS-MAPK-pathway by miR-181a mimics represents a novel promising therapeutic approach for AML and possibly for other RAS-driven cancers.

SPARC promotes leukemic cell growth and predicts acute myeloid leukemia outcome.

Aberrant expression of the secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) gene, which encodes a matricellular protein that participates in normal tissue remodeling, is associated with a variety of diseases including cancer, but the contribution of SPARC to malignant growth remains controversial. We previously reported that SPARC was among the most upregulated genes in cytogenetically normal acute myeloid leukemia (CN-AML) patients with gene-expression profiles predictive of unfavorable outcome, such as mutations in isocitrate dehydrogenase 2 (IDH2-R172) and overexpression of the oncogenes brain and acute leukemia, cytoplasmic (BAALC) and v-ets erythroblastosis virus E26 oncogene homolog (ERG). In contrast, SPARC was downregulated in CN-AML patients harboring mutations in nucleophosmin (NPM1) that are associated with favorable prognosis. Based on these observations, we hypothesized that SPARC expression is clinically relevant in AML. Here, we found that SPARC overexpression is associated with adverse outcome in CN-AML patients and promotes aggressive leukemia growth in murine models of AML. In leukemia cells, SPARC expression was mediated by the SP1/NF-κB transactivation complex. Furthermore, secreted SPARC activated the integrin-linked kinase/AKT (ILK/AKT) pathway, likely via integrin interaction, and subsequent ß-catenin signaling, which is involved in leukemia cell self-renewal. Pharmacologic inhibition of the SP1/NF-κB complex resulted in SPARC downregulation and leukemia growth inhibition. Together, our data indicate that evaluation of SPARC expression has prognosticative value and SPARC is a potential therapeutic target for AML.

RUNX1 mutations are associated with poor outcome in younger and older patients with cytogenetically normal acute myeloid leukemia and with distinct gene and MicroRNA expression signatures.

PURPOSE: To determine the association of RUNX1 mutations with therapeutic outcome in younger and older patients with primary cytogenetically normal acute myeloid leukemia (CN-AML) and with gene/microRNA expression signatures. PATIENTS AND METHODS: Younger (< 60 years; n = 175) and older (≥ 60 years; n = 225) patients with CN-AML treated with intensive cytarabine/anthracycline-based first-line therapy on Cancer and Leukemia Group B protocols were centrally analyzed for RUNX1 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene/microRNA expression profiles were derived using microarrays. RESULTS: RUNX1 mutations were found in 8% and 16% of younger and older patients, respectively (P = .02). They were associated with ASXL1 mutations (P < .001) and inversely associated with NPM1 (P < .001) and CEBPA (P = .06) mutations. RUNX1-mutated patients had lower complete remission rates (P = .005 in younger; P = .006 in older) and shorter disease-free survival (P = .058 in younger; P < .001 in older), overall survival (P = .003 in younger; P < .001 in older), and event-free survival (P < .001 for younger and older) than RUNX1 wild-type patients. Because RUNX1 mutations were more common in older patients and almost never coexisted with NPM1 mutations, RUNX1 mutation-associated expression signatures were derived in older, NPM1 wild-type patients and featured upregulation of genes normally expressed in primitive hematopoietic cells and B-cell progenitors, including DNTT, BAALC, BLNK, CD109, RBPMS, and FLT3, and downregulation of promoters of myelopoiesis, including CEBPA and miR-223. CONCLUSION: RUNX1 mutations are twice as common in older than younger patients with CN-AML and negatively impact outcome in both age groups. RUNX1-mutated blasts have molecular features of primitive hematopoietic and lymphoid progenitors, potentially leading to novel therapeutic approaches.

Ascorbic acid induces apoptosis in adult T-cell leukemia.

BACKGROUND: Adult T-cell leukemia (ATL) is an acute malignancy of activated T-cells caused by the human T-cell lymphotrophic virus type-1 (HTLV-1). MATERIALS AND METHODS: The effects of non-cytotoxic concentrations of ascorbic acid (AA) were evaluated against HTLV-1 positive and negative cells. The effect of AA on apoptosis and proliferation was evaluated by cell cycle analysis. The role of p53, p21 Bax and Bcl-2a on cell cycle modulation and apoptosis was also assessed. The anti-proliferative effects were tested by determining the changes in the expression of transforming growth factors (TGF-alpha, TGF-beta1 and TGF-beta2). RESULTS: Ascorbic acid was found to reduce the proliferation of cells and induce apoptosis by the modulation of p53, p21, Bcl-2 and Bax. CONCLUSION: The results of this study show the anti-proliferative effects of AA against leukemic cells.