The BLM helicase is a new therapeutic target in multiple myeloma involved in replication stress survival and drug resistance.

Multiple myeloma (MM) is a hematologic cancer characterized by accumulation of malignant plasma cells in the bone marrow. To date, no definitive cure exists for MM and resistance to current treatments is one of the major challenges of this disease. The DNA helicase BLM, whose depletion or mutation causes the cancer-prone Bloom's syndrome (BS), is a central factor of DNA damage repair by homologous recombination (HR) and genomic stability maintenance. Using independent cohorts of MM patients, we identified that high expression of BLM is associated with a poor outcome with a significant enrichment in replication stress signature. We provide evidence that chemical inhibition of BLM by the small molecule ML216 in HMCLs (human myeloma cell lines) leads to cell cycle arrest and increases apoptosis, likely by accumulation of DNA damage. BLM inhibition synergizes with the alkylating agent melphalan to efficiently inhibit growth and promote cell death in HMCLs. Moreover, ML216 treatment re-sensitizes melphalan-resistant cell lines to this conventional therapeutic agent. Altogether, these data suggest that inhibition of BLM in combination with DNA damaging agents could be of therapeutic interest in the treatment of MM, especially in those patients with high BLM expression and/or resistance to melphalan.

PIM2 kinase has a pivotal role in plasmablast generation and plasma cell survival, opening up novel treatment options in myeloma.

The differentiation of B cells into plasmablasts (PBs) and then plasma cells (PCs) is associated with extensive cell reprogramming and new cell functions. By using specific inhibition strategies (including a novel morpholino RNA antisense approach), we found that early, sustained upregulation of the proviral integrations of Moloney virus 2 (PIM2) kinase is a pivotal event during human B-cell in vitro differentiation and then continues in mature normal and malignant PCs in the bone marrow. In particular, PIM2 sustained the G1/S transition by acting on CDC25A and p27Kip1 and hindering caspase 3-driven apoptosis through BAD phosphorylation and cytoplasmic stabilization of p21Cip1. In PCs, interleukin-6 triggered PIM2 expression, resulting in antiapoptotic effects on which malignant PCs were particularly dependent. In multiple myeloma, pan-PIM and myeloid cell leukemia-1 (MCL1) inhibitors displayed synergistic activity. Our results highlight a cell-autonomous function that links kinase activity to the newly acquired secretion ability of the PBs and the adaptability observed in both normal and malignant PCs. These findings should finally prompt the reconsideration of PIM2 as a therapeutic target in multiple myeloma.

Targeting Cellular Iron Homeostasis with Ironomycin in Diffuse Large B-cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy. Although more than half of patients with DLBCL achieve long-term remission, the majority of remaining patients succumb to the disease. As abnormal iron homeostasis is implicated in carcinogenesis and the progression of many tumors, we searched for alterations in iron metabolism in DLBCL that could be exploited to develop novel therapeutic strategies. Analysis of the iron metabolism gene expression profile of large cohorts of patients with DLBCL established the iron score (IS), a gene expression-based risk score enabling identification of patients with DLBCL with a poor outcome who might benefit from a suitable targeted therapy. In a panel of 16 DLBCL cell lines, ironomycin, a promising lysosomal iron-targeting small molecule, inhibited DLBCL cell proliferation at nanomolar concentrations compared with typical iron chelators. Ironomycin also induced significant cell growth inhibition, ferroptosis, and autophagy. Ironomycin treatment resulted in accumulation of DNA double-strand breaks, delayed progression of replication forks, and increased RPA2 phosphorylation, a marker of replication stress. Ironomycin significantly reduced the median number of viable primary DLBCL cells of patients without major toxicity for nontumor cells from the microenvironment and presented low toxicity in hematopoietic progenitors compared with conventional treatments. Significant synergistic effects were also observed by combining ironomycin with doxorubicin, BH3 mimetics, BTK inhibitors, or Syk inhibitors. Altogether, these data demonstrate that a subgroup of high-risk patients with DLBCL can be identified with the IS that can potentially benefit from targeting iron homeostasis. SIGNIFICANCE: Iron homeostasis represents a potential therapeutic target for high-risk patients with DLBCL that can be targeted with ironomycin to induce cell death and to sensitize tumor cells to conventional treatments.

G9a/GLP targeting in MM promotes autophagy-associated apoptosis and boosts proteasome inhibitor-mediated cell death.

Multiple myeloma (MM) is an (epi)genetic highly heterogeneous plasma cell malignancy that remains mostly incurable. Deregulated expression and/or genetic defects in epigenetic-modifying enzymes contribute to high-risk disease and MM progression. Overexpression of the histone methyltransferase G9a was reported in several cancers, including MM, correlating with disease progression, metastasis, and poor prognosis. However, the exact role of G9a and its interaction partner G9a-like protein (GLP) in MM biology and the underlying mechanisms of action remain poorly understood. Here, we report that high G9a RNA levels are associated with a worse disease outcome in newly diagnosed and relapsed MM patients. G9a/GLP targeting using the specific G9a/GLP inhibitors BIX01294 and UNC0638 induces a G1-phase arrest and apoptosis in MM cell lines and reduces primary MM cell viability. Mechanistic studies revealed that G9a/GLP targeting promotes autophagy-associated apoptosis by inactivating the mTOR/4EBP1 pathway and reducing c-MYC levels. Moreover, genes deregulated by G9a/GLP targeting are associated with repressive histone marks. G9a/GLP targeting sensitizes MM cells to the proteasome inhibitors (PIs) bortezomib and carfilzomib, by (further) reducing mTOR signaling and c-MYC levels and activating p-38 and SAPK/JNK signaling. Therapeutic treatment of 5TGM1 mice with BIX01294 delayed in vivo MM tumor growth, and cotreatment with bortezomib resulted in a further reduction in tumor burden and a significantly prolonged survival. In conclusion, we provide evidence that the histone methyltransferases G9a/GLP support MM cell growth and survival by blocking basal autophagy and sustaining high c-MYC levels. G9a/GLP targeting represents a promising strategy to improve PI-based treatment in patients with high G9a/GLP levels.

Maternal embryonic leucine zipper kinase is a novel target for diffuse large B cell lymphoma and mantle cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are among the most aggressive B cell non-Hodgkin lymphomas. Maternal embryonic leucine zipper kinase (MELK) plays a role in cancer cell cycle progression and is associated with poor prognosis in several cancer cell types. In this study, the role of MELK in DLBCL and MCL and the therapeutic potential of MELK targeting is evaluated. MELK is highly expressed in DLBCL and MCL patient samples, correlating with a worse clinical outcome in DLBCL. Targeting MELK, using the small molecule OTSSP167, impaired cell growth and survival and induced caspase-mediated apoptosis in the lymphoma cells. Western blot analysis revealed that MELK targeting decreased the phosphorylation of FOXM1 and the protein levels of EZH2 and several mitotic regulators, such as Cdc25B, cyclin B1, Plk-1, and Aurora kinases. In addition, OTSSP167 also sensitized the lymphoma cells to the clinically relevant Bcl-2 inhibitor venetoclax by strongly reducing Mcl1 levels. Finally, OTSSP167 treatment of A20-inoculated mice resulted in a significant prolonged survival. In conclusion, targeting MELK with OTSSP167 induced strong anti-lymphoma activity both in vitro and in vivo. These findings suggest that MELK could be a potential new target in these aggressive B cell malignancies.

Phenotypic Characterization of Diffuse Large B-Cell Lymphoma Cells and Prognostic Impact.

Multiparameter flow cytometry (MFC) is a fast and cost-effective technique to evaluate the expression of many lymphoid markers in mature B-cell neoplasms, including diffuse large B cell lymphoma (DLBCL), which is the most frequent non-Hodgkin lymphoma. In this study, we first characterized by MFC the expression of 27 lymphoid markers in 16 DLBCL-derived cell lines to establish a robust algorithm for their authentication. Then, using the expression profile in DLBCL samples of the genes encoding B lymphoid markers that are routinely investigated by MFC, we built a gene expression-based risk score, based on the expression level of BCL2, BCL6, CD11c, and LAIR1, to predict the outcome of patients with DLBCL. This risk score allowed splitting patients in four risk groups, and was an independent predictor factor of overall survival when compared with the previously published prognostic factors. Lastly, to investigate the potential correlation between BCL2, BCL6, CD11c, and LAIR1 protein level and resistance to treatment, we investigated the response of the 16 DLBCL cell lines to cyclophosphamide, etoposide, doxorubicin, and gemcitabine. We found a correlation between BCL6 overexpression and resistance to etoposide. These results show the interest of MFC for the routine characterization of DLBCL cells and tumors samples for research and diagnostic/prognostic purposes.

The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma.

BACKGROUND: The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL. METHODS: The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot. RESULTS: We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax. CONCLUSION: We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL.

An epigenetic regulator-related score (EpiScore) predicts survival in patients with diffuse large B cell lymphoma and identifies patients who may benefit from epigenetic therapy.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma and shows considerable clinical and biological heterogeneity. Much research is currently focused on the identification of prognostic markers for more specific patients' risk stratification and on the development of therapeutic approaches to improve the long-term outcome. Epigenetic alterations are involved in various cancers, including lymphoma. Interestingly, epigenetic alterations are reversible and drugs to target some of them have been developed. In this study, we demonstrated that the gene expression profile of epigenetic regulators has a prognostic value in DLBCL and identified pathways that could be involved in DLBCL poor outcome. We then designed a new risk score (EpiScore) based on the gene expression level of the epigenetic regulators DNMT3A, DOT1L, SETD8. EpiScore was predictive of overall survival in DLBCL and allowed splitting patients with DLBCL from two independent cohorts (n = 414 and n = 69) in three groups (high, intermediate and low risk). EpiScore was an independent predictor of survival when compared with previously described prognostic factors, such as the International Prognostic Index (IPI), germinal center B cell and activated B cell molecular subgroups, gene expression-based risk score (GERS) and DNA repair score. Immunohistochemistry analysis of DNMT3A in 31 DLBCL samples showed that DNMT3A overexpression (>42% of positive tumor cells) correlated with reduced overall and event-free survival. Finally, an HDAC gene signature was significantly enriched in the DLBCL samples included in the EpiScore high-risk group. We conclude that EpiScore identifies high-risk patients with DLBCL who could benefit from epigenetic therapy.

Diagnostic and prognostic value of soluble CD14 subtype (Presepsin) for sepsis and community-acquired pneumonia in ICU patients.

BACKGROUND: The soluble CD14 subtype, Presepsin, appears to be an accurate sepsis diagnostic marker, but data from intensive care units (ICUs) are scarce. This study was conducted to evaluate the diagnostic and prognostic value of Presepsin in ICU patients with severe sepsis (SS), septic shock (SSh) and severe community-acquired pneumonia (sCAP). METHODS: Presepsin and procalcitonin (PCT) levels were determined for patients at admission to ICU. Four groups have been differentiated: (1) absence or (2) presence of systemic inflammatory response syndrome, (3) SS or (4) SSh; and 2 groups, among the patients admitted for acute respiratory failure: absence or presence of sCAP. Biomarkers were tested for diagnosis of SS, SSh and sCAP and for prediction of ICU mortality. RESULTS: One hundred and forty-four patients were included: 44 SS and 56 SSh. Plasma levels of Presepsin and PCT were significantly higher in septic than in non-septic patients and in SSh as compared to others. The sepsis diagnostic accuracy of Presepsin was not superior to that of PCT (AUC: 0.75 vs 0.80). In the 72/144 patients admitted for acute respiratory failure, the capability of Presepsin to diagnose sCAP was significantly better than PCT. Presepsin levels were also predictive of ICU mortality in sepsis and in sCAP patients. CONCLUSION: Plasma levels of Presepsin were useful for the diagnosis of SS, SSh and sCAP and may predict ICU mortality in these patients.