In this issue of Blood, Wu et al(1) describe a novel yet critical transcriptional factor network that confers resistance to immunomodulatory imide drugs (IMiDs) in T-cell lymphomas (TCLs). They identify a pivotal codependence of Ikaros (IKZF1) and zinc finger protein 91 (ZFP91) in IMiD-resistant TCLs, thereby revealing an acquired mechanism of resistance to IMiD treatment. Furthermore, their study provides an attractive therapeutic strategy that uses more potent next-generation degraders targeting IKZF1 and ZFP91 for treating patients with TCL.

Anaplastic large cell lymphoma (ALCL) is a CD30-positive non-Hodgkin’s T‑cell lymphoma. Despite the implementation of CD30 antibody–drug conjugate-targeted therapy into front-line treatment regimens, the prognosis of some subtypes of the disease remains unsatisfactory. In the relapsed/refractory setting, effective second-line treatment options are still lacking. However, it has been reported that blockade of direct downstream targets of activator protein‑1 (AP-1) transcription factors, which are highly dysregulated in ALCL, results in complete and sustained remission in late-stage relapsed/refractory anaplastic lymphoma kinase (ALK)-positive ALCL patients. Moreover, it has been identified that involvement of the BATF3/AP‑1 module promotes lymphomagenesis via oncogenic BATF3/IL-2/IL-2R signaling through hyperphosphorylation of ERK1/2, STAT1, and STAT5 in ALCL cells regardless of their ALK status. Therefore, targeting BATF3/IL-2/IL-2R signaling may represent a novel therapeutic alternative for ALCL patients.

We recently reported that miR-146a is differentially expressed in ALK+ and ALK− anaplastic large cell lymphoma (ALCL). In this study, the downstream targets of miR-146a in ALK+ ALCL were investigated by transcriptome analysis, identifying CD147 as potential target gene. Because CD147 is differentially expressed in ALK+ ALCL versus ALK− ALCL and normal T cells, this gene emerged as a strong candidate for the pathogenesis of this tumor. Here we demonstrate that CD147 is a direct target of miR-146 and contributes to the survival and proliferation of ALK+ ALCL cells in vitro and to the engraftment and tumor growth in vivo in an ALK+ ALCL-xenotransplant mouse model. CD147 knockdown in ALK+ ALCL cells resulted in loss of monocarboxylate transporter 1 (MCT1) expression, reduced glucose consumption and tumor growth retardation, as demonstrated by [ 18 F]FDG-PET/MRI analysis. Investigation of metabolism in vitro and in vivo supported these findings, revealing reduced aerobic glycolysis and increased basal respiration in CD147 knockdown. In conclusion, our findings indicate that CD147 is of vital importance for ALK+ ALCL to maintain the high energy demand of rapid cell proliferation, promoting lactate export, and tumor growth. Furthermore, CD147 has the potential to serve as a novel therapeutic target in ALK+ ALCL, and warrants further investigation.

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Systemic anaplastic large cell lymphoma (sALCL) encompasses two distinct clinical entities of T-cell non-Hodgkin lymphoma: anaplastic lymphoma kinase-positive (ALK+) ALCL and ALK-negative (ALK-) ALCL. These entities are characterized by either the presence or absence of an ALK translocation. It has been reported that ALK+ ALCL has a better prognosis compared to ALK-, with a 5-year overall survival (OS) of 70–80% versus 40–60%, respectively, [1,2,3]. Furthermore, more than 30% of ALK+ ALCL patients relapse [4, 5]. Despite the distinction between the two sALCL subtypes, frontline treatment for adults is similar and is based on CHOP or CHOEP, instead pediatric ALCL patients are mainly treated following the ALCL99 protocol [6,7,8]. Whilst high-throughput genomic studies in sALCL have shown recurrent genetic alterations, their association with outcome has not been fully investigated [9,10,11,12,13]. In this study, the mutational landscape of sALCL patient tumors was investigated to discover potential biomarkers that may improve risk stratification and patient management.

Anaplastic Large Cell Lymphoma (ALCL) is a T-cell malignancy predominantly driven by a hyperactive Anaplastic Lymphoma Kinase (ALK) fusion protein. ALK inhibitors such as crizotinib provide alternatives to standard chemotherapy with reduced toxicity and side effects. Children with lymphomas driven by NPM1-ALK fusion proteins achieved an objective response rate to ALK inhibition therapy of 54-90% in clinical trials. However, a subset of patients progress within the first 3 months of treatment. The mechanism for the development of ALK inhibitor resistance is unknown. Through genome-wide CRISPR activation and knockout screens in ALCL cell lines combined with RNA-seq data derived from ALK inhibitor relapsed patient tumors, we show that resistance to ALK inhibition by crizotinib in ALCL can be driven by aberrant upregulation of IL10RA. Elevated IL10RA expression rewires the STAT3 signaling pathway bypassing otherwise critical phosphorylation by NPM1-ALK. IL10RA expression does not correlate with response to standard chemotherapy in pediatric patients suggesting that combination of crizotinib with chemotherapy could prevent ALK-inhibitor resistance-specific relapse. Trials registered as NCT01979536/NCT02034981/UMIN000028075.

BACKGROUND: Systemic Anaplastic Large Cell Lymphoma (sALCL) is comprised of two distinct T-cell non-Hodgkin lymphoma entities: ALK-positive (ALK+) ALCL and ALK-negative (ALK-) ALCL. These entities are characterized by either the presence of absence of an ALK-translocation. It has been reported that ALK+ ALCL has a better prognosis compared to ALK- ALCL, with a 5-year overall survival (OS) of 70-80% versus 15-45%, respectively. Furthermore, more than 25% of ALK+ ALCL patients undergo relapse. sALCL is a genetically heterogeneous disease whose genomic characterization has been improved through the implementation of high-throughput technologies. Despite this, the prognostic value of somatic mutations has been poorly described. Here we present the analysis of genetic aberrations of sALCL, shedding light on disease pathogenesis, novel diagnostic biomarkers and prognostic markers for the detection of refractory and/or relapsed patients which could hold clinical implications. METHODS: Formalin-fixed paraffin-embedded (FFPE) and/or fresh frozen tissue and related clinical information for 82 sALCL patients (47 ALK+ and 35 ALK-) were obtained after written informed consent from 5 centres across Europe. Using deep targeted DNA sequencing, the entire coding region of 275 selected genes (QIAseq Targeted DNA - Human Comprehensive Cancer Panel, Qiagen, Germany) was investigated in our retrospective cohort of patient' samples as well as 6 ALCL cell lines (Karpas-299, SU-DHL-1, DEL, SR786, FEPD and MAC2a). The average depth achieved across all the sequenced samples was approximately 2000x allowing the detection of aberrations with low allele frequencies (<10%) not usually detectable by other methods (e.g. Sanger sequencing or Whole Exome Sequencing). Moreover, we explored the clonal expansion in sALCL relapsed patients comparing the mutational status in matched samples at both diagnosis and relapse (n=4). RESULTS: ALK- patients in our cohort have a worse prognosis than ALK+, whereby the 7y-OS was 45.1% and 73.2%, respectively, although the 7 year progression free survival (PFS) was comparable at 57.1% for ALK+ and 42.4% for ALK- patients. As previously reported, ALK+ patients were significantly younger in our series than ALK- patients with an average age of 23.5 and 55.2 years, respectively. Among the 275 genes sequenced, 148 (53.8%) genes harbor at least one mutation throughout the entire patient and cell line cohort; almost 1/3 of mutated genes were shared between ALK- and ALK+ ALCL patients. On average 4.2 mutations per patient were detected in ALK- ALCL, a higher level than observed in ALK+ samples (2.7), most likely due to the oncogenic-driving role of the ALK-translocation in this group. Seventy-two out of 82 (88%) patients carried at least one single mutation within the genes analysed in our panel. The most recurrent genes mutated were TP53 in both sALCL categories (16% of sALCL patients), followed by LRP1B being prevalently mutated in ALK+ disease (15% of sALCL patients). As expected, STAT3 and JAK1 were mutated solely in the ALK- ALCL cohort and represent the most commonly mutated genes in this group. Prognostically, the most recurrent genes mutated in patients with a dismal outcome (dead and/or relapsed patients) were TP53, STAT3, EPHA5, JAK1, LRP1B, KMT2D, PRDM1 and SOCS1. Comparing samples at diagnosis versus relapse highlighted two key findings: firstly, the clonal expansion of TP53 mutated clones in relapsed ALCL patients and secondly, the acquired mutations in EPHA5 which were only detected at relapse. These data suggest their possible use as biomarkers associated with clonal evolution. CONCLUSION: This study provides information regarding the genetic landscape in sALCL across 275 select genes. We have confirmed the importance of known frequently mutated genes (STAT3 and JAK1) and describe novel recurrently mutated genes (LRP1B, EPHA5 and KMT2D). Mutations in STAT3 were more recurrent in ALK- ALCL patients with shorter OS, and clones harboring mutated TP53 and EPHA5 were detectable more often in relapsed sALCL thereby suggesting a possible driving role. Disclosures Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

The Activator Protein-1 (AP-1) transcription factor (TF) family, composed of a variety of members including c-JUN, c-FOS and ATF, is involved in mediating many biological processes such as proliferation, differentiation and cell death. Since their discovery, the role of AP-1 TFs in cancer development has been extensively analysed. Multiple in vitro and in vivo studies have highlighted the complexity of these TFs, mainly due to their cell-type specific homo- or hetero-dimerization resulting in diverse transcriptional response profiles. However, as a result of the increasing knowledge of the role of AP-1 TFs in disease, these TFs are being recognized as promising therapeutic targets for various malignancies. In this review, we focus on the impact of deregulated expression of AP-1 TFs in CD30-positive lymphomas including Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma.

Transcription factor AP-1 is constitutively activated and IRF4 drives growth and survival in ALK+ and ALK– anaplastic large cell lymphoma (ALCL). Here we demonstrate high-level BATF and BATF3 expression in ALCL. Both BATFs bind classical AP-1 motifs and interact with in ALCL deregulated AP-1 factors. Together with IRF4, they co-occupy AP-1-IRF composite elements, differentiating ALCL from non-ALCL. Gene-specific inactivation of BATFs, or global AP-1 inhibition results in ALCL growth retardation and/or cell death in vitro and in vivo. Furthermore, the AP-1-BATF module establishes TH17/group 3 innate lymphoid cells (ILC3)-associated gene expression in ALCL cells, including marker genes such as AHR, IL17F, IL22, IL26, IL23R and RORγt. Elevated IL-17A and IL-17F levels were detected in a subset of children and adolescents with ALK+ ALCL. Furthermore, a comprehensive analysis of primary lymphoma data confirms TH17–, and in particular ILC3-skewing in ALCL compared with PTCL. Finally, pharmacological inhibition of RORC as single treatment leads to cell death in ALCL cell lines and, in combination with the ALK inhibitor crizotinib, enforces death induction in ALK+ ALCL. Our data highlight the crucial role of AP-1/BATFs in ALCL and lead to the concept that some ALCL might originate from ILC3.

TYK2 is a member of the JAK family of tyrosine kinases that is involved in chromosomal translocation-induced fusion proteins found in anaplastic large cell lymphomas (ALCL) that lack rearrangements activating the anaplastic lymphoma kinase (ALK). Here we demonstrate that TYK2 is highly expressed in all cases of human ALCL, and that in a mouse model of NPM-ALK-induced lymphoma, genetic disruption of Tyk2 delays the onset of tumors and prolongs survival of the mice. Lymphomas in this model lacking Tyk2 have reduced STAT1 and STAT3 phosphorylation and reduced expression of Mcl1 , a pro-survival member of the BCL2 family. These findings in mice are mirrored in human ALCL cell lines, in which TYK2 is activated by autocrine production of IL-10 and IL-22 and by interaction with specific receptors expressed by the cells. Activated TYK2 leads to STAT1 and STAT3 phosphorylation, activated expression of MCL1 and aberrant ALCL cell survival. Moreover, TYK2 inhibitors are able to induce apoptosis in ALCL cells, regardless of the presence or absence of an ALK-fusion. Thus, TYK2 is a dependency that is required for ALCL cell survival through activation of MCL1 expression. TYK2 represents an attractive drug target due to its essential enzymatic domain, and TYK2-specific inhibitors show promise as novel targeted inhibitors for ALCL.

Topoisomerase II inhibitor ellipticine effectively suppressed the growth of human non-small-cell-lung-cancer (NSCLC) epithelial cells. Previously, we reported the drug activity was consummated through parallel nucleus migration of p53 and Akt in A549 cells. While inducing cell death, the drug activity was proved related to autophagy through phosphorylated Akt at S473. In addition, ellipticine induced cytotoxicity in p53-null H1299 cells with stable expression of ectopic p53. In this work, we further demonstrated that dominant-negative Akt S473A or p53 shRNA inhibited ellipticine-mediated translocalization of p53 and Akt and attenuated apoptotic cell death in A549 cells. The presence of p53 predates ellipticine-mediated apoptotic cell death, assists in nucleus translocation of phosphorylated Akt and activation of autophagy pathway. Growth inhibition through collaborating p53 and phosphorylated Akt473 in lung epithelial cancer cells provided a new perspective of the topoisomerase inhibitor as an effective cancer therapy agent.

Topoisomerase II inhibitor ellipticine and its analogues were reported as promising antitumor agents. We have reported previously that ellipticine induced nuclear translocation of p53 and Akt followed by autophagic apoptosis in human non‐small cell lung cancer cells A549. The effect can be reverted by autophagy inhibitors. In this work, we further demonstrated that transfection of p53 siRNA suppressed ellipticine‐induced apoptosis by blocking Akt nuclear translocation. In addition, introduction of either point‐mutated Akt473 or Akt308 inhibited p53 translocation as indicated by confocal microscopy and western blotting. On the other hand, in p53‐null H1299 cells with stably expressed wild type p53, cells become sensitive to ellipticine treatment accompanied with nucleus translocation of p53 and Akt. In addition, as cells were pretreated with autophagy inhibitor before being treated with ellipticine, Akt phosphorylation was suppressed with decreased apoptotic cell death. We concluded nuclear translocation of phosphorylated Akt is essential in leading to ellipticine‐induced cell death in human non‐small cell lung cancer cells.The work is supported by grants from the National Science Council, Taiwan (NSC100‐2311‐B‐003‐003) and National Taiwan Normal University (99‐D3)