Molecular mechanisms and pathogenetic role of BCL6 deregulation in diffuse large B-cell lymphomas — clinical and therapeutic implications

Deregulacja onkogenu BCL6 jest najczęstszym zaburzeniem molekularnym w chłoniaku rozlanymz dużych komórek B (DLBCL) u dorosłych. Gen BCL6 po raz pierwszy sklonowanoz translokacji chromosomalnych dotyczących 3q27. Gen ten koduje białko należące do rodzinyrepresorów transkrypcyjnych charakteryzujących się obecnością domeny BTB/POZ oraz palcówcynkowych. Dimeryzacja BCL6 powoduje rekrutację korepresorów, które odpowiadają zazróżnicowane biologiczne funkcje tego białka. Ekspresja BCL6 w limfocytach B jest niezbędnado rozpoczęcia reakcji germinalnej, w trakcie której komórki B intensywnie proliferująi wskutek procesów edycji sekwencji genów immunoglobulinowych zwiększają powinowactwoprzeciwciał do antygenu. W obrębie centrum germinalnego BCL6 kontroluje ekspresję genów,których represja zwiększa tolerancję na fizjologiczne uszkodzenia DNA wynikające ze specyfikireakcji germinalnej, reguluje progresję cyklu komórkowego, aktywację limfocytów oraz ichróżnicowanie. Wyciszenie ekspresji BCL6 jest wymagane do zakończenia reakcji germinalneji dalszego różnicowania limfocytów w komórki pamięci lub plazmocyty. Konstytutywna ekspresjaBCL6 wskutek zaburzeń strukturalnych lub mechanizmów czynnościowych uniemożliwiaróżnicowanie limfocytów, sprzyja akumulacji dodatkowych zaburzeń genetycznych i indukujepowstanie DLBCL. Z tego względu inhibicja aktywności BCL6 może stanowić racjonalnycel terapeutyczny w chłoniakach z deregulacją tego onkogenu i czynnościowo zależnych od jegoekspresji. Poznanie molekularnych zależności między strukturą i funkcją białka BCL6 pozwoliłona podjęcie prób projektowania molekuł, które mogłyby specyficznie rozrywać interakcjeBCL6 z określonymi zestawami korepresorów i swoiście blokować jego określone funkcje.Niniejszy artykuł stanowi przegląd doniesień dotyczących roli BCL6 w genezie DLBCL orazobecnego stanu zaawansowania badań dotyczących celowanych inhibitorów BCL6.BCL6 is the most frequently deregulated oncogene in diffuse large B-cell lymphoma (DLBCL),the most common lymphoma in adults. BCL6 gene was first cloned from recurringchromosomal translocations involving 3q27. BCL6 encodes a member of the BTB/POZ zincfinger family of transcriptional repressors. Upon dimerization, BCL6 recruits distinct sets ofcorepressors that mediate its biological activity. BCL6 expression in B-cells is required forformation of germinal centers (GC). Within the GC, BCL6 controls the expression of multiplegenes that increase the tolerance to the physiological DNA breaks required for immunoglobulinaffinity maturation, regulate cell cycle progression, B-cell activation and differentiation.Downregulation of BCL6 is required for B-cells to undergo further differentiation to memorycells or plasma cells. Constitutive expression of BCL6 due to structural or functionalabnormalities in humans and mice inhibits B-cell differentiation and fosters acquisition ofadditional lesions, leading to DLBCL. For these reasons, BCL6 represents a rationaltherapeutic target in DLBCL with deregulated expression of this oncogene and reliant on itscontinuous activity. Dissection of molecular structure-function relationships of the BCL6protein allowed to design molecules specifically targeting this transcription factor and blockingits function. Herein, we review recent advances in understanding the role of BCL6 inlymphomagenesis and approaches to its therapeutic targeting

Deregulation of MYC transcription factor in lymphoid tumors — molecular, pathogenetic, clinical and therapeutic implications

Czynnik transkrypcyjny MYC jest jednym z najczęściej deregulowanych onkogenów w ludzkichnowotworach. Gen MYC koduje białko należące do rodziny czynników transkrypcyjnychzawierających motyw suwaka leucynowego, kontrolujące szeroki zakres genów odpowiedzialnychza przyspieszoną proliferację, metabolizm komórkowy, wzrost, angiogenezę, zdolność dotworzenia przerzutów, niestabilność genetyczną, potencjał odtwórczy komórek macierzystychoraz zredukowaną zdolność różnicowania. W mysich modelach rozwinięcie pełnego fenotypunowotworów zainicjowanych przez MYC wymaga dodatkowych zmian, takich jak: mutacjeścieżki p53, mutacje TCF3, aktywność PI3K oraz zaburzenia białek z rodziny BCL2.W ludzkich nowotworach komórek B rearanżacje MYC obejmujące region 8q24 oraz genyimmunoglobulinowe są cechą charakterystyczną chłoniaka Burkitta, ale występują równieżw innych nowotworach. W chłoniaku rozlanym z dużych komórek B MYC jest trzecim, poBCL6 i BCL2, najczęściej deregulowanym onkogenem. Proste rearanżacje MYC/IGH są rzadkospotykane w tych nowotworach i częściej dotyczą genów łańcuchów lekkich lub genównieimmunoglobulinowych oraz towarzyszą złożonym kariotypom. Najczęstszą cytogenetycznąnieprawidłowością towarzyszącą MYC jest t(14;18) dotycząca BCL2 lub rzadziej rearanżacjeBCL6. Takie chłoniaki double/triple-hit, zawierające rearanżacje BCL2 i/lub BCL6 wrazz nieprawidłowościami MYC, często mają nietypowe cechy morfologiczne i immunofenotypoweoraz charakteryzują się agresywnym przebiegiem. Biorąc pod uwagę szeroki zakres funkcjiMYC, inhibicja jego aktywności może być racjonalną strategią terapeutyczną w leczeniu chłoniakówwykazujących nadekspresję tego onkogenu. W poniższym artykule dokonano przeglądupatogenetycznych mechanizmów związanych z deregulacją MYC w ludzkich nowotworachz komórek B oraz potencjału terapeutycznego inhibitorów tego czynnika transkrypcyjnego.MYC transcription factor is one of the most frequently deregulated oncogenes in humanmalignancies. It encodes a leucine zipper transcription factor that controls a broad spectrum ofcellular genes responsible for enhanced proliferation, cellular metabolism, growth, vasculogenesis,metastasis, genomic instability, stem cell self-renewal and reduced differentiation. Inmouse models, full-blown phenotype of MYC-driven tumors requires cooperation with otherlesions, including p53 pathway mutations, TCF3 mutations, PI3K activity and BCL2 familyabnormalities. In human B-cell tumors, MYC rearrangements involving 8q24 region andimmunoglobulin genes are hallmark of Burkitt lymphoma, but also occur in other lymphoidmalignancies. In diffuse large B-cell lymphoma, MYC is the third, after BCL6 and BCL2,most commonly deregulated oncogene. Simple MYC/IGH rearrangements in these tumors areuncommon and more frequently involve light chain or non-IG partners and exist in the settingof complex karyotypes. The most common cytogenetic abnormality accompanying MYC ist(14;18) involving BCL2 and less frequently BCL6. Such “double/triple hit” B-cell tumorsharboring BCL2 and/or BCL6 rearrangements concurrent with MYC lesions frequently exhibitatypical morphologic and/or immunophenotypic features and particularly aggressive clinicalbehavior. Given the broad range of MYC functions, inhibition of MYC activity might bea rational therapeutic strategy in lymphoid tumors expressing this oncogene. Several approachesfor pharmaceutical intervention have been suggested. Herein, we review the molecularpathogenetic mechanisms associated with MYC deregulation in human B-cell tumors and itsimplications for such targeted therapies

Nanoparticles for Immune Cytokine TRAIL-Based Cancer Therapy

The immune cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received significant attention as a cancer therapeutic due to its ability to selectively trigger cancer cell apoptosis without causing toxicity in vivo. While TRAIL has demonstrated significant promise in preclinical studies in mice as a cancer therapeutic, challenges including poor circulation half-life, inefficient delivery to target sites, and TRAIL resistance have hindered clinical translation. Recent advances in drug delivery, materials science, and nanotechnology are now being exploited to develop next-generation nanoparticle platforms to overcome barriers to TRAIL therapeutic delivery. Here, we review the design and implementation of nanoparticles to enhance TRAIL-based cancer therapy. The platforms we discuss are diverse in their approaches to the delivery problem and provide valuable insight into guiding the design of future nanoparticle-based TRAIL cancer therapeutics to potentially enable future translation into the clinic. ©2018 American Chemical Society.Cancer Center Support (core) Grant (P30-CA14051)Ruth L. Kirschstein National Research Service Award from the NIH (F32CA200351)Burroughs Wellcome Fund (No. 1015145

B Cell Restricted Expression of Mutated IKZF3 modulates BCR Signaling and Homing Pathways in a Mouse Model of CLL

International audienceMutation in IKZF3 (AIOLOS), a lymphoid transcription factor with a key role in B cell development and function, has been identified as a putative driver of chronic lymphocytic leukemia (CLL) through large-scale WES studies of CLL patients. Prevalent in ~3% CLLs and associated with fludarabine resistance, mutated IKZF3 has been detected uniquely as a hotspot mutation (L162R), localized within the DNA binding domain. The functional effects exerted by this mutation on lymphomagenesis remain unexplored, and were the subject of the current study. We generated a B-cell restricted knock-in mouse line by crossing mice with the Ikzf3 mutant floxed allele with Cd19-Cre animals (i.e. Cre recombinase expression under the Cd19 promoter), and established cohorts carrying either heterozygous mutant-Ikzf3 (Ikzf3Het), homozygous mutant-Ikzf3 (Ikzf3Homo) or wild-type Ikzf3 (Ikzf3WT). We thereafter monitored the animals for leukemia development by serial flow cytometry analyses of peripheral blood. Notably, we detected appearance of ~10-50% clonal B220+CD5+Igk+in 8 of 30 (27%) of the Ikzf3Het mice by 20 months of age, but not in age-matched Ikzf3WTmice (n=30). The cohort of Ikzf3Homo mice has been monitored thus far for 18 months, with identification already of one of 30 (3%) with leukemia with a ~15 months onset. Across animals with CLL-like disease, the pattern of disease localization was consistently observed as detected in the spleen but not in the bone marrow, suggesting preferential homing of Ikzf3-mutant cells to the splenic microenvironment. To dissect the mechanisms underlying Ikzf3-mediated leukemogenesis, we asked whether presence of mutated-Ikzf3 was associated with changes in B cell transcriptional programs in young mice without disease. We performed RNA-sequencing using splenic B cells from Ikzf3Het, Ikzf3Homo or Ikzf3wt animals (3 mice/group, 3-month old). The most striking changes were identified between Ikzf3Homo to Ikzf3wt mice, with ~1400 differentially expressed genes in Ikzf3Homocells compared to Ikzf3wt (>2 fold, adjusted p<0.05). Within this Ikzf3Homo-specific signature, we identified genes regulating B cell positioning within the light zone of germinal centers (GCs, p<0.0001) and BCR signaling pathway members (p<0.05), by gene set enrichment analysis (GSEA). The enhanced activation of the BCR pathway was further confirmed by western blot analysis of splenic B cells stimulated with anti-IgM, with Ikzf3Homo and Ikzf3het mice showing upregulation of phosphorylation in SYK, AKT and ERK after BCR engagement, as compared to Ikzf3WT. Allo-immunization with the T-cell dependent antigen sheep red blood cells (SRBC) for 10 days elicited a higher rate of germinal centers (GC) formation in Ikzf3Homo mice than Ikzf3WT (p<0.01, ANOVA with Tukey's correction), further indicating an enhanced response to BCR stimulation in presence of the mutation. Overall, phenotypic changes of Ikzf3het appeared less pronounced than Ikzf3Homo, in young mice. To further define the functional effects of mutant-Ikzf3 in transcriptional regulation, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using anti-AIOLOS antibody in Ikzf3WT and Ikzf3Homo B cells. While we observed no significant changes in the conserved DNA binding motif or the genomic localization of AIOLOS binding sites, stronger AIOLOS binding at the transcription start sites (TSS) was detected in Ikzf3Homo versus Ikzf3WTcells (p=1.3747x10-5, Student's t-test). These results indicate an enhanced binding of mutated AIOLOS to DNA which may in turn dysregulate gene expression. As an example, upregulation of the chemokine receptor Cxcr4 in Ikzf3Homo B cells was associated with an enrichment of mutant AIOLOS at its promoter region. Functionally, we confirmed that such upregulation was associated with increased migration of Ikzf3Homo cells to SDF-1 in vitro, in a transwell-based chemotaxis assay (p<0.01, ANOVA with Tukey's correction). Overall, this novel model provides evidence of an oncogenic role of IKZF3 mutation in CLL. Ikzf3 mutation induces broad transcriptional and functional changes associated with dysregulation of BCR signaling and homing mechanisms, which may favor disease initiation and progression in vivo. Comparative analyses between Ikzf3-mutant murine CLLs and IKZF3-mutant primary samples are ongoing, to further refine the relevance of Ikzf3 in B cell function and lymphomagenesis. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding

IKZF3 Overexpression Phenocopies Gain-of-Function Mutation in Chronic Lymphocytic Leukemia

A hotspot mutation within the DNA-binding domain of IKZF3 (IKZF3-L162R) has been identified as a putative driver in chronic lymphocytic leukemia (CLL); however, its functional effects are unknown. We recently confirmed its role as a CLL driver in a B cell-restricted conditional knock-in model. IKZF3 mutation altered mature B cell development and signaling capacity, and induced CLL-like disease in elderly mice (~40% penetrance). Moreover, we found IKZF3-L162R acts as a gain-of-function mutation, altering DNA binding specificity and target selection of IKZF3, and resulting in overexpression of multiple B-cell receptor (BCR) genes. Consistent with the murine data, RNA-sequencing analysis showed that human CLL cells with mut-IKZF3 [n=4] have an enhanced signature of BCR-signaling gene expression compared to WT-IKZF3 [n=6, all IGHV unmutated] (p<0.001), and also exhibited general upregulation of key BCR-signaling regulators. These results confirm the role of IKZF3 as a master regulator of BCR-signaling gene expression, with the mutation contributing to overexpression of these genes. While mutation in IKZF3 has a clear functional impact on a cardinal CLL-associated pathway, such as BCR signaling, we note that this driver occurs only at low frequency in patients (~3%). Because somatic mutation represents but one mechanism by which a driver can alter a cellular pathway, we examined whether aberrant expression of IKZF3 could also yield differences in BCR-signaling gene expression. We have observed expression of the IKZF3 gene to be variably dysregulated amongst CLL patients through re-analysis of transcriptomic data from two independent cohorts of human CLL (DFCI, Landau et al., 2014; ICGC, Ferreira et al., 2014). We thus examined IKZF3 expression and BCR-signaling gene expression, or the 'BCR score' (calculated as the mean expression of 75 BCR signaling-associate genes) in those cohorts (DFCI cohort, n=107; ICGC cohort, n=274). Strikingly, CLL cells with higher IKZF3 expression (defined as greater than median expression) had higher BCR scores than those with lower IKZF3 expression (<median) (p=0.0015 and p<0.0001, respectively). These findings were consistent with the notion that IKZF3 may act as a broad regulator of BCR signaling genes, and that IKZF3 overexpression, like IKZF3 mutation, may provide fitness advantage. In support of this notion, our re-analysis of a gene expression dataset of 107 CLL samples (Herold Leukemia 2011) revealed that higher IKZF3 expression associated with poorer prognosis and worse overall survival (P=0.035). We previously reported that CLL cells with IKZF3 mutation appeared to increase in cancer cell fraction (CCF) with resistance to fludarabine-based chemotherapy (Landau Nature 2015). Instances of increase in mut-IKZF3 CCF upon treatment with the BCR-signaling inhibitor ibrutinib have been reported (Ahn ASH 2019). These studies together suggest an association of IKZF3 mutation with increased cellular survival following either chemotherapy or targeted treatment. To examine whether higher expression of IKZF3 was associated with altered sensitivity to ibrutinib, we performed scRNA-seq analysis (10x Genomics) of two previously treatment-naïve patients undergoing ibrutinib therapy (paired samples, baseline vs. Day 220). We analyzed an average of 11,080 cells per patient (2000 genes/cell). Of note, following ibrutinib treatment, remaining CLL cells expressed higher levels of IKZF3 transcript compared to pretreatment baseline (both p<0.0001), whereas no such change was observed in matched T cells (n ranging between 62 to 652 per experimental group, p>0.05), suggesting that cells with high expression of IKZF3 were selected by ibrutinib treatment. Moreover, we showed that ibrutinib treatment resulted in consistent upregulation of BCR-signaling genes (e.g., CD79B, LYN, GRB2, FOS, RAC1, PRKCB and NFKBIA) (n ranging between 362 to 1374 per experimental group, all p<0.0001), which were likewise activated by mutant IKZF3. Altogether, these data imply that IKZF3 mutation or overexpression may influence upregulation of BCR-signaling genes and enhance cellular fitness even during treatment with BCR-signaling inhibitors. We highlight our observation that IKZF3 mutation appears to be phenocopied by elevated IKZF3 expression, and suggest that alterations in mRNA or protein level that mimic genetic mutations could be widespread in human cancers. Disclosures Kipps: Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding