Inactivation of the PRDM1/BLIMP1 gene in diffuse large B cell lymphoma

PR domain containing 1 with zinc finger domain (PRDM1)/B lymphocyte–induced maturation protein 1 (BLIMP1) is a transcriptional repressor expressed in a subset of germinal center (GC) B cells and in all plasma cells, and required for terminal B cell differentiation. The BLIMP1 locus lies on chromosome 6q21-q22.1, a region frequently deleted in B cell lymphomas, suggesting that it may harbor a tumor suppressor gene. We report here that the BLIMP1 gene is inactivated by structural alterations in 24% (8 out of 34) activated B cell–like diffuse large cell lymphoma (ABC-DLBCL), but not in GC B cell–like (n = 0/37) or unclassified (n = 0/21) DLBCL. BLIMP1 alterations included gene truncations, nonsense mutations, frameshift deletions, and splice site mutations that generate aberrant transcripts encoding truncated BLIMP1 proteins. In all cases studied, both BLIMP1 alleles were inactivated by deletions or mutations. Furthermore, most non–GC type DLBCL cases (n = 20/26, 77%) lack BLIMP1 protein expression, despite the presence of BLIMP1 mRNA. These results indicate that a sizable fraction of ABC-DLBCL carry an inactive BLIMP1 gene, and suggest that the same gene is inactivated by epigenetic mechanisms in an additional large number of cases. These findings point to a role for BLIMP1 as a tumor suppressor gene, whose inactivation may contribute to lymphomagenesis by blocking post–GC differentiation of B cells toward plasma cells

Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency.

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.We thank Maria Stella Scalzo for technical support, Dr Emanuela Colombo for kindly providing MEFs that lack NPM1 (MEF NPM−/−p53−/−) and control fibroblasts (MEF p53−/−), Dr Guido Serini for the use of his confocal microscopy unit at the Candiolo Cancer Institute—IRCCS, Torino, Italy. We also thank Ariad Pharmaceutical, Pfizer, Astellas and Novartis that kindly provided all drugs used in this study. This work was supported by the Regione Lombardia (ID14546A) and Fondazione Berlucchi Onlus Grant 2014 (to CGP), and by grants FP7 ERC-2009-StG (Proposal No. 242965—‘Lunely’); Associazione Italiana per la Ricerca sul Cancro (AIRC) Grant IG-12023; Koch Institute/DFCC Bridge Project Fund; Ellison Foundation Boston; Worldwide Cancer Research Association (former AICR) grant 12-0216; the Grant for Oncology Innovation by Merck-Serono and R01 CA196703-01 (to RC).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/onc.2015.45

Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells

Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme that targets immunoglobulin (Ig) genes to initiate class switch recombination (CSR) and somatic hypermutation (SHM)(1). Through off-target activity, however, AID has a much broader impact on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in lymphoma development and progression(2). AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation(3). The phosphatidylinositol 3-kinase (PI3K) δ pathway plays a key role in AID regulation by suppressing its expression in B cells(4). Novel drugs for leukemia or lymphoma therapy such as idelalisib, duvelisib or ibrutinib block PI3Kδ activity directly or indirectly(5–8), potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation (SHM) and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both these effects were completely abrogated in AID deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumors in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IgH and AID off-target sites in human chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased SHM in AID off-targets. In summary, we show that PI3Kδ or BTK inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism, an effect that should be carefully considered as such inhibitors are administered for years to patients

Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system

Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM+ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab′ fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production

Redundant and nonredundant roles for Cdc42 and Rac1 in lymphomas developed in NPM-ALK transgenic mice

Increasing evidence suggests that Rho family GTPases could have a critical role in the biology of T-cell lymphoma. In ALK-rearranged anaplastic large cell lymphoma (ALCL), a specific subtype of T-cell lymphoma, the Rho family GTPases Cdc42 and Rac1 are activated by the ALK oncogenic activity. In vitro studies have shown that Cdc42 and Rac1 control rather similar phenotypes of ALCL biology such as the proliferation, survival, and migration of lymphoma cells. However, their role and possible redundancy in ALK-driven lymphoma development in vivo are still undetermined. We genetically deleted Cdc42 or Rac1 in a mouse model of ALK-rearranged ALCL to show that either Cdc42 or Rac1 deletion impaired lymphoma development, modified lymphoma morphology, actin filament distribution, and migration properties of lymphoma cells. Cdc42 or Rac1 deletion primarily affected survival rather than proliferation of lymphoma cells. Apoptosis of lymphoma cells was equally induced following Cdc42 or Rac1 deletion, was associated with upregulation of the proapoptotic molecule Bid, and was blocked by Bcl2 overexpression. Remarkably, Cdc42/Rac1 double deletion, but not Cdc42 or Rac1 single deletions, completely prevented NPM-ALK lymphoma dissemination in vivo. Thus, Cdc42 and Rac1 have nonredundant roles in controlling ALK-rearranged lymphoma survival and morphology but are redundant for lymphoma dissemination, suggesting that targeting both GTPases could represent a preferable therapeutic option for ALCL treatment

A Radiological Approach to Evaluate Bone Graft Integration in Reconstructive Surgeries

(1) Background: Bone tissue engineering is a promising tool to develop new smart solutions for regeneration of complex bone districts, from orthopedic to oral and maxillo-facial fields. In this respect, a crucial characteristic for biomaterials is the ability to fully integrate within the patient body. In this work, we developed a novel radiological approach, in substitution to invasive histology, for evaluating the level of osteointegration and osteogenesis, in both qualitative and quantitative manners. (2) SmartBone®, a composite xeno-hybrid bone graft, was selected as the base material because of its remarkable effectiveness in clinical practice. Using pre- and post-surgery computed tomography (CT), we built 3D models that faithfully represented the patient’s anatomy, with special attention to the bone defects. (3) Results: This way, it was possible to assess whether the new bone formation respected the natural geometry of the healthy bone. In all cases of the study (four dental, one maxillo-facial, and one orthopedic) we evaluated the presence of new bone formation and volumetric increase. (4) Conclusion: The newly established radiological protocol allowed the tracking of SmartBone® effective integration and bone regeneration. Moreover, the patient’s anatomy was completely restored in the defect area and functionality completely rehabilitated without foreign body reaction or inflammation