Anaplastic Large Cell Lymphoma Cells Research Articles

Establishment and characterization of TK-ALCL1: a novel NPM-ALK-positive anaplastic large-cell lymphoma cell line.

TK-ALCL1, a novel anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALK+ ALCL) cell line, was established from the primary tumor site of a 59-year-old Japanese male patient. The immune profile of TK-ALCL1 corresponds to that seen typically in primary ALCL cells, i.e., positive for ALK, CD30, EMA, and CD4, but negative for CD2, CD3, CD5, CD8a, and EBV-related antigens. The rearrangement of the T cell receptor-gamma locus shows that TK-ALCL1 is clonally derived from T-lineage lymphoid cells. FISH and RT-PCR analysis revealed that TK-ALCL1 has the nucleophosmin (NPM)-ALK fusion transcript, which is typical for ALK+ ALCL cell lines. When TK-ALCL1 was subcutaneously inoculated into 6-week-old BALB/c Rag2-/-/Jak3-/- (BRJ) mice, it formed tumor masses within 4-6weeks. Morphological, immunohistochemical, and molecular genetic investigations confirmed that the xenograft and the original ALCL tumor were identical. The ALK inhibitors Alectinib and Lorlatinib suppressed proliferation in a dose-dependent manner. Thus, TK-ALCL1 provides a useful in vitro and in vivo model for investigation of the biology of ALK+ ALCL and of novel therapeutic approaches targeting ALK.

HELLS regulates transcription in T-cell lymphomas by reducing unscheduled R-loops and by facilitating RNAPII progression.

Chromatin modifiers are emerging as major determinants of many types of cancers, including Anaplastic Large Cell Lymphomas (ALCL), a family of highly heterogeneous T-cell lymphomas for which therapeutic options are still limited. HELLS is a multifunctional chromatin remodeling protein that affects genomic instability by participating in the DNA damage response. Although the transcriptional function of HELLS has been suggested, no clues on how HELLS controls transcription are currently available. In this study, by integrating different multi-omics and functional approaches, we characterized the transcriptional landscape of HELLS in ALCL. We explored the clinical impact of its transcriptional program in a large cohort of 44 patients with ALCL. We demonstrated that HELLS, loaded at the level of intronic regions of target promoters, facilitates RNA Polymerase II (RNAPII) progression along the gene bodies by reducing the persistence of co-transcriptional R-loops and promoting DNA damage resolution. Importantly, selective knockdown of HELLS sensitizes ALCL cells to different chemotherapeutic agents, showing a synergistic effect. Collectively, our work unveils the role of HELLS in acting as a gatekeeper of ALCL genome stability providing a rationale for drug design.

Abstract 4320: Interrogation of autophagy inhibition in anaplastic large cell lymphoma (ALCL)

Abstract The objectives of this study are to evaluate if anaplastic lymphoma kinase (ALK) inhibition activates autophagy in ALK+ ALCL cells and to determine if that activation can be mitigated using autophagy inhibition. ALCL is an aggressive T-cell non-Hodgkin lymphoma that often presents as advanced stage disease. With the addition of crizotinib or brentuximab vedotin to the ALCL99 chemotherapy backbone, the recently reported Children’s Oncology Group ANHL12P1 study demonstrated a 2-year event-free survival for ALCL patients of 79%. Thus, even with multi-agent chemotherapy combined with targeted therapy, improved therapeutic efficacy is needed. Autophagy is activated when mTORC1 is inhibited, and as ALK signaling is known to promote mTORC1 activity, ALK inhibition is hypothesized to activate autophagy. If present, ALK inhibitor-induced autophagy activation would represent a novel therapeutic opportunity targetable with emerging autophagy inhibitors. An ALK+ ALCL cell line, Karpas 299, was treated with drug combinations for 3 hours, lysates prepared, and proteins separated by molecular weight using standard procedures. ALK inhibitor target engagement was assessed by western blot using phospho-ALKY1604 intensity in 9-point dose response curves. Target engagement of autophagy inhibition (ULK-101 directed against ULK1/2 and hydroxychloroquine (HCQ) targeting the lysosome) was assessed by western blot using phospho-ATG14S29 and LC3B-II intensity in 6-point dose response curves. Cell viability was evaluated using a luminescent CellTiter-Glo assay. Karpas299 cells resistant to crizotinib at 50nM (KCrizR50) were generated by gradually increasing crizotinib concentration in the media until cells grew at a rate similar to parental cells. Crizotinib, ceritinib, and lorlatinib all effectively inhibit ALK phosphorylation in Karpas 299 cells with low nanomolar potency. ULK-101 effectively inhibited pATG14S29 at 5 µM and HCQ effectively inhibited autophagic flux, as evidenced by increased LC3B-II intensity, at 20 µM. When treated with the lowest ALK inhibitor dose needed to achieve complete pALK inhibition (IC99), pATG14 intensity increased representing increased autophagic activity. When combined with ALK inhibition, ULK-101 effectively decreased pATG14S29, blocking the ALK inhibitor-induced autophagy activation. Decreased viability in Karpas 299 cells was observed after crizotinib, ceritinib and lorlatinib treatments with nanomolar potency. KCrizR50 cells had a potency higher than parental Karpas 299 cells for all three ALK inhibitors. ALK inhibition activates autophagy in ALCL cells and autophagy activation is effectively mitigated through concurrent autophagy inhibition. ALK inhibitor resistance in Karpas 299 cells will be further evaluated to determine whether autophagy contributes to the resistant phenotype. Citation Format: Katie L. Buelow, Stephanie Celano, Gregory Poole, Katie Martin, Jeffrey MacKeigan, David J. Hoogstra. Interrogation of autophagy inhibition in anaplastic large cell lymphoma (ALCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4320.

STAT3 couples activated tyrosine kinase signaling to the oncogenic core transcriptional regulatory circuitry of anaplastic large cell lymphoma

Anaplastic large cell lymphoma (ALCL) is an aggressive, CD30+ Tcell lymphoma of children and adults. ALK fusion transcripts or mutations in the JAK-STAT pathway are observed in most ALCL tumors, but the mechanisms underlying tumorigenesis are not fully understood. Here, we show that dysregulated STAT3 in ALCL cooccupies enhancers with master transcription factors BATF3, IRF4, and IKZF1 to form a core regulatory circuit that establishes and maintains the malignant cell state in ALCL. Critical downstream targets of this network in ALCL cells include the protooncogene MYC, which requires active STAT3 to facilitate high levels of MYC transcription. The core autoregulatory transcriptional circuitry activity is reinforced by MYC binding to the enhancer regions associated with STAT3 and each of the core regulatory transcription factors. Thus, activation of STAT3 provides the crucial link between aberrant tyrosine kinase signaling and the core transcriptional machinery that drives tumorigenesis and creates therapeutic vulnerabilities in ALCL.

SGN-35C: A Novel CD30-Directed Antibody-Drug Conjugate for the Treatment of Lymphomas

SGN-35C is an antibody drug conjugate composed of an anti-CD30 antibody conjugated to a camptothecin-derived topoisomerase 1 (TOP1) inhibitor payload. SGN-35C was designed to leverage the antibody backbone from brentuximab vedotin (BV) and the novel mechanism of action of camptothecin-derived ADCs. Here, we evaluated the mechanism of action, cytotoxicity, and safety in in vitro and in vivo models. To characterize the mechanism of action of SGN-35C in vitro and in vivo experiments were performed in CD30-positive anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma (HL) cell lines, and versions of these cell lines that are resistant to BV. SGN-35C binding, internalization, induction of DNA damage, cell cycle analysis, and in vitro cytotoxicity were evaluated. To assess if payload released in CD30-positive cells could kill adjacent CD30-negative cells, an in vitro bystander assay of CD30-positive and CD30-negative co-cultures was developed. The in vivo antitumor activity of SGN-35C was evaluated in animals bearing subcutaneous lymphoma models. SGN-35C antitumor activity was evaluated in a xenograft model of heterogeneous CD30 expression consisting of a mixture of a BV-resistant Karpas-299 cell line mixed with parental Karpas-299 cells to assess bystander activity in vivo. The tolerability of SGN-35C was assessed in a non-human primate model. SGN-35C binding to CD30-positive cells was similar to the unconjugated anti-CD30 antibody and demonstrated internalization into CD30-expressing cells. Treatment of cells with SGN-35C for 24 hours led to an increase of the DNA damage marker phospho-γH2A.X, which induced an increase in the fraction of cells in S-phase, consistent with the mechanism of camptothecin-based TOP1 inhibitors. Cytotoxicity was observed in ALCL and HL cell lines treated with SGN-35C, including an ALCL cell line resistant to BV. In a mixture of CD30-positive and CD30-negative cells, SGN-35C treatment induced cytotoxicity of the CD30-negative cells, demonstrating that SGN-35C can elicit bystander activity. A single dose of SGN-35C led to significant tumor growth inhibition in animals bearing lymphoma xenografts, including a BV-resistant model and a model of heterogeneous CD30 expression, demonstrating that SGN-35C can elicit bystander activity in vivo. SGN-35C demonstrates a tolerability profile in non-human primates that is consistent with camptothecins. A Phase 1, first-in-human study is planned to evaluate the safety and anti-tumor activity of SGN-35C in lymphoid malignancies.

Pre-Clinical Activity of Navitoclax in TCR-Driven and Non-ALCL Mature T-Cell Lymphomas

Background: In contrast to many B-cell lymphomas that highly express BCL-2, BCL-2 expression is observed in <50% of most mature T-cell lymphoma (MTCL) subtypes, although a clinical trial investigating venetoclax in BCL-2 positive relapsed/refractory PTCL is ongoing (NCT03552692). In contrast, BCL-xL and/or MCL-1 are expressed in the majority of MTCL. Consistent with these findings, MCL-1 expressing anaplastic large cell lymphomas (ALCL) and typical (non-ETP) T-ALL, which highly express BCL-xL, have been shown by BH3 profiling to be dependent upon MCL-1 or BCL-xL, respectively. Among the more common MTCL subtypes, including cutaneous T-cell lymphomas (CTCL), the extent to which BCL-2 family members are a therapeutic vulnerability is uncertain. However, we have previously demonstrated that antigen- and costimulatory receptor signaling promote the growth and survival of these lymphomas and confers resistance to chemotherapy, and in conventional (non-malignant) T cells, these same signals upregulate BCL-xL, which is required for their clonal expansion and escape from cell death following T-cell activation. Therefore, given the increasing availability of selective BH3 mimetics and the need for improved therapeutic strategies, we were motivated to examine BCL-2 family member dependencies in these lymphomas. Methods: Peptides generated from BH3-only proteins and selective BH3 mimetics were utilized to perform “BH3 profilng” in MTCL. Cell viability upon exposure to BH3 mimetics was determined using standard approaches. Results: To pre-clinically assess BCL-2 family member dependencies and the mitochondrial apoptotic pathway in T-cell lymphoproliferative neoplasms, we performed BH3 profiling in 17 cell lines. Peptides derived from BIM and PUMA, which are promiscuous, were utilized to assess the extent to which cells are “primed” for apoptosis. Differences in apoptotic priming were not observed across subtypes, and most cell lines examined were highly primed. We next examined the dependence on specific antiapoptotic proteins by BH3 profiling using BAD (antagonizes BCL-2, BCL-xL, and BCL-w), HRK (antagonizes BCL-xL), MS1 (antagonizes MCL-1), and FS1 (antagonizes A1/BFL-1) peptides. BCL-2 dependence was not observed in the T-cell lymphoma cell lines we examined. However, and in stark contrast, most non-ALCL cell lines were BCL-xL dependent, whereas ALCL cell lines were largely MCL-1 dependent. In an effort to further confirm these results, and examine the extent to which peptide-based BH3 profiling predicts sensitivity to selective BH3 mimetics in MTCL, cell lines were treated with venetoclax, A1155463 (a BCL-xL selective antagonist), navitoclax (a BCL-2/BCL-xL antagonist), or S63845 (MCL-1 antagonist), and cell viability determined. Consistent with peptide-based BH3 profiling, all cell lines examined were resistant to venetoclax. ALCL cell lines were largely MCL-1 dependent, whereas the non-ALCL cell lines examined were largely BCL-xL dependent. Therefore, we examined BCL-xL and MCL-1 expression in these cells and observed a strong association between BCL-xL expression and both cytochrome c release in response to HRK peptide and cell viability in the presence of A1155463. A similar association was observed between MCL-1 expression and sensitivity to MS1 and S63845. Transcriptionally profiled MTCL specimens were stratified by MTCL classification (ALCL vs. non-ALCL) and in keeping with the data obtained using cell lines increased BCL-xL expression was observed in non-ALCL MTCL subtypes. Given the observed association between BCL-xL expression and vulnerability to BCL-xL antagonists, we sought to examined mechanisms that promote its expression. Both TCR signaling and CD28 costimulation are exploited by non-ALCL subtypes. We examined the extent to which TCR/CD28 stimulation regulates BCL-xL transcription. In MTCL specimens, a significant increase in BCL-xL transcription and sensitization to Bcl-xL antagonism were observed upon TCR/CD28 stimulation. A TCR-dependent GEM model was utilized, demonstrating that these MTCL are Bcl-xL dependent and sensitive to navitoclax. Conclusions: These findings suggest that BCL-xL is a therapeutic vulnerability for MTCL subsets, particularly non-ALCL subtypes that are TCR dependent, and provide a robust pre-clinical rationale for future studies investigating navitoclax in these lymphomas.

A Combination of Alectinib and DNA-Demethylating Agents Synergistically Inhibits Anaplastic-Lymphoma-Kinase-Positive Anaplastic Large-Cell Lymphoma Cell Proliferation.

The recent evolution of molecular targeted therapy has improved clinical outcomes in several human malignancies. The translocation of anaplastic lymphoma kinase (ALK) was originally identified in anaplastic large-cell lymphoma (ALCL) and subsequently in non-small cell lung carcinoma (NSCLC). Since ALK fusion gene products act as a driver of carcinogenesis in both ALCL and NSCLC, several ALK tyrosine kinase inhibitors (TKIs) have been developed. Crizotinib and alectinib are first- and second-generation ALK TKIs, respectively, approved for the treatment of ALK-positive ALCL (ALK+ ALCL) and ALK+ NSCLC. Although most ALK+ NSCLC patients respond to crizotinib and alectinib, they generally relapse after several years of treatment. We previously found that DNA-demethylating agents enhanced the efficacy of ABL TKIs in chronic myeloid leukemia cells. Moreover, aberrant DNA methylation has also been observed in ALCL cells. Thus, to improve the clinical outcomes of ALK+ ALCL therapy, we investigated the synergistic efficacy of the combination of alectinib and the DNA-demethylating agent azacytidine, decitabine, or OR-2100 (an orally bioavailable decitabine derivative). As expected, the combination of alectinib and DNA-demethylating agents synergistically suppressed ALK+ ALCL cell proliferation, concomitant with DNA hypomethylation and a reduction in STAT3 (a downstream target of ALK fusion proteins) phosphorylation. The combination of alectinib and OR-2100 markedly altered gene expression in ALCL cells, including that of genes implicated in apoptotic signaling, which possibly contributed to the synergistic anti-ALCL effects of this drug combination. Therefore, alectinib and OR-2100 combination therapy has the potential to improve the outcomes of patients with ALK+ ALCL.

An Approach to Nodal T- and NK-Cell Lymphomas—A Systemic Review

AbstractT-cell lymphomas are rare neoplasms that have complex pathology. The multiparameter approach has been recommended by World Health Organization (WHO) for the classification of T-cell lymphomas taking into account morphology, immunophenotype, genetics, and clinical features. This also includes division established on the possible cell-of-origin (COO) from T regulatory or T-follicular helper (TFH) cells. The recent WHO-HAEM5 has classified entities as precursor T-lymphoblastic neoplasms, mature T-cell neoplasms, Epstein-Barr virus (EBV)-related T- and NK/T-cell lymphomas, and tumor-like lesions with T-cells predominance. Distinct entities have been recognized within the anaplastic large cell lymphoma (ALCL) family founded on the status of anaplastic lymphoma kinase (ALK) gene rearrangement: ALK-positive and molecularly heterogeneous ALK-negative. The family of lymphomas arising from TFH cells consists of three distinct nodal TFH cell lymphoma entities: angioimmunoblastic-type, follicular-type, and not otherwise specified. These three entities show significant clinical and immunophenotypic overlap. The cases that do not qualify for ALCL or nodal TFH cell lymphomas are labelled as peripheral T-cell lymphomas-not otherwise specified after ruling out nodal EBV-positive T- and NK- cell lymphoma. The new category termed tumor-like lesions with T cell predominance has a high chance to be misdiagnosed as lymphoma. This category includes entities such as Kikuchi-Fujimoto disease, indolent T-lymphoblastic proliferation, and autoimmune lymphoproliferative syndrome. For pathologists, diagnosing nodal T-cell lymphomas may be thought-provoking due to their broad histopathologic spectrum that mimics reactive as well as other neoplastic processes. This review provides a comprehensive diagnostic criterion of the most commonly encountered nodal T-cell and NK cell lymphomas in day-to-day training and an algorithmic approach.

MiR-146a-5p enrichment in small-extracellular vesicles of relapsed pediatric ALCL patients promotes macrophages infiltration and differentiation

Anaplastic large cell lymphoma (ALCL) is a CD30-positive lymphoma accounting for 20% of all pediatric T-cell lymphomas. Current first line treatment can cure most of ALCL patients but 10–30% of them are resistant or relapse. In this context, liquid biopsy has the potential to help clinicians in disease screening and treatment response monitoring.Small-RNA-sequencing analysis performed on plasma small-extracellular vesicles (s-EVs) from 20 pediatric anaplastic lymphoma kinase positive (ALK + ) ALCL patients at diagnosis revealed a specific miRNAs cargo in relapsed patients compared to non-relapsed, with seven miRNAs enriched in s-EVs of relapsed patients. MiR-146a-5p and miR-378a-3p showed a negative prognostic impact both in univariate and multivariate analysis, possibly representing, together with let-7 g-5p, a miRNA panel for the early identification of high-risk patients.Among them, miR-146a-5p is known to modulate tumor supporting-M2 macrophages differentiation, but the role of these cells in pediatric ALK + ALCL is still unknown. To elucidate the role of miR-146a-5p and M2 macrophages in pediatric ALCL disease, THP-1-derived macrophages were treated with s-EVs from ALK + ALCL cell lines, showing increased miR-146a-5p intracellular expression, migrating capability and M2-markers CD163 and Arginase-1 upregulation. In turn, conditioned media from M2 macrophages or miR-146a-5p-transfected THP-1 increased ALCL cells' aggressive features and were enriched in interleukin-8.Overall, these data suggest a role of miR-146a-5p in promoting macrophage infiltration and M2-like polarization in ALCL. Our findings incite further investigation on the role of M2 macrophages in ALCL aggressiveness and dissemination, also considering the novel treatment options targeting tumor associated macrophages.

Aberrant expression of GOLM1 protects ALK+ anaplastic large cell lymphoma from apoptosis by enhancing BCL-XL stability.

Golgi membrane protein 1 (GOLM1) is aberrantly expressed in many types of solid tumors and contributes to cancer development; however, its role in hematopoietic and lymphoid neoplasms remains unknown. Here, we report that GOLM1 was significantly upregulated in anaplastic large cell lymphoma (ALCL), particularly in anaplastic lymphoma kinase-positive (ALK+) ALCL. Mechanistically, the expression of GOLM1 was induced by nucleophosmin-ALK in both ALK-transformed T cells and ALCL cell lines through AKT/mTOR pathway. Knockdown of GOLM1 expression led to a reduction in the growth and viability of ALCL cells with increased spontaneous apoptosis, whereas ectopic expression of GOLM1 protected ALCL cells from apoptosis induced by staurosporine treatment. Moreover, GOLM1 directly interacted with B-cell lymphoma-extra large protein (a crucial anti-apoptosis regulator) and significantly prolonged its stability. Introduction of GOLM1 promoted ALK+ ALCL cells colony formation invitro and tumor growth in a murine xenograft model. Taken together, our findings demonstrate, to our knowledge, for the first time that GOLM1 plays a critical role in suppressing apoptosis and promoting the progression of ALK+ ALCL and provide evidence that GOLM1 is a potential biomarker and therapeutic target in ALK-induced hematological malignancies.

Plasma small‐extracellular vesicles enriched in miR‐122‐5p promote disease aggressiveness in pediatric anaplastic large‐cell lymphoma

Plasma small‐extracellular vesicles enriched in miR‐122‐5p promote disease aggressiveness in pediatric anaplastic large‐cell lymphoma

Regulation of CD45 phosphatase by oncogenic ALK in anaplastic large cell lymphoma.

Anaplastic Large Cell Lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma frequently driven by the chimeric tyrosine kinase NPM-ALK, generated by the t (2,5)(p23;q35) translocation. While ALK+ ALCL belongs to mature T cell lymphomas, loss of T cell identity is observed in the majority of ALCL secondary to a transcriptional and epigenetic repressive program induced by oncogenic NPM-ALK. While inhibiting the expression of T cell molecules, NPM-ALK activates surrogate TCR signaling by directly inducing pathways downstream the TCR. CD45 is a tyrosine phosphatase that plays a central role in T cell activation by controlling the TCR signaling and regulating the cytokine responses through the JAK/STAT pathway and exists in different isoforms depending on the stage of T-cell maturation, activation and differentiation. ALK+ ALCL cells mainly express the isoform CD45RO in keeping with their mature/memory T cell phenotype. Because of its regulatory effect on the JAK/STAT pathway that is essential for ALK+ ALCL, we investigated whether CD45 expression was affected by oncogenic ALK. We found that most ALK+ ALCL cell lines express the CD45RO isoform with modest CD45RA expression and that NPM-ALK regulated the expression of these CD45 isoforms. Regulation of CD45 expression was dependent on ALK kinase activity as CD45RO expression was increased when NPM-ALK kinase activity was inhibited by treatment with ALK tyrosine kinase inhibitors (TKIs). Silencing ALK expression through shRNA or degradation of ALK by the PROTAC TL13-112 caused upregulation of CD45RO both at mRNA and protein levels with minimal changes on CD45RA, overall indicating that oncogenic ALK downregulates the expression of CD45. CD45 repression was mediated by STAT3 as demonstrated by ChIP-seq data on ALCL cells treated with the ALK-TKI crizotinib or cells treated with a STAT3 degrader. Next, we found that knocking-out CD45 with the CRISPR/Cas9 system resulted in increased resistance to ALK TKI treatment and CD45 was down-regulated in ALCL cells that developed resistance in vitro to ALK TKIs. Overall, these data suggest that CD45 expression is regulated by ALK via STAT3 and acts as a rheostat of ALK oncogenic signaling and resistance to TKI treatment in ALCL.

PP2A Inhibitor LB100 Enhances the Sensitivity of NPM-ALK+ALCL to Crizotinib and Reverses the Resistance of ALCL to Crizotinib

Anaplastic large cell lymphoma (ALCL) is a rare and aggressive non-Hodgkin's lymphoma commonly discovered in children and adolescents. Despite the high remission rates in ALCL achieved by conventional chemotherapy based on conventional CHOP (cyclophosphamide, adriamycin, vincristine, prednisone), relapse and drug resistance occur in more than 40% of patients. The clinical use of the ALK inhibitor crizotinib in most relapsed refractory NPM-ALK+ ALCL in recent years has demonstrated effective treatment, but crizotinib resistance and intolerance due to the corresponding side effects still occur in 30% of cases. This urgently requires us to find new treatments to enhance the sensitivity of crizotinib as well as to overcome the resistance of crizotinib in ALCL. PP2A has been considered a tumor suppressor gene in the past, but with better understanding, it is two-sided, playing different roles in various types of tumors. In addition, it has been found that PP2A is closely related to drug sensitivity and resistance. LB100, a novel water-soluble PP2A inhibitor, is gaining attention because of its significantly higher safety profile than traditional PP2A inhibitors. It has potent effects on tumor inhibition and could increase tumor sensitivity to radiotherapy. In this study, we first established a crizotinib-resistant NPM-ALK+ ALCL cell model by exposing a highly sensitive NPM-ALK-positive ALCL cell line to continuously increasing doses of crizotinib until resistance developed. The PP2A inhibitor LB100 was then applied to normal and resistant cell lines to explore changes in their biological activity. We found that LB100 significantly inhibited the viability of normal and drug-resistant ALCL cell lines and was more sensitive in drug-resistant lines. In addition, a combination of LB100 and crizotinib was applied in normal and resistant cell lines to analyze the effect of LB100 on sensitization to crizotinib and reversal of drug resistance. LB100 significantly enhanced the killing effect of crizotinib in normal tumor cell lines and partially reverses crizotinib resistance in resistant lines. Western blot and immunofluorescence showed that LB100 leads to increased cellular G1 phase block by increasing cellular DNA damage, which in turn enhances the sensitivity of crizotinib. The above experiments showed that LB100, as an effective inhibitor of PP2A, can significantly increase the effect of crizotinib against normal NPM-ALK+ ALCL, inhibit the growth of crizotinib-resistant strains, and partially restore the sensitivity of crizotinib, which is expected to be a new therapeutic tool for ALCL. In addition, simultaneous targeting of PP2A and ALK has a very high potential in overcoming ALK inhibitor resistance in ALCL and could be a new therapeutic strategy to overcome drug resistance.

TCL-318 Evaluation of the Antitumor Activity of Amiloride in Peripheral T-Cell Lymphomas Using In-Vitro Models.

Peripheral T-cell lymphomas (PTCL) have an aggressive clinical behavior and poor response to treatment; therefore, the search for new drugs is required. We have previously demonstrated the antitumor activity of amiloride in multiple myeloma. This prompted us to investigate the possibility of a cytotoxic effect of amiloride in this kind of T-cell lymphoma. To evaluate the antitumor activity of amiloride in different PTCL using in-vitro assays and identify the possible mechanisms involved. Two anaplastic large-cell lymphoma (ALCL) cell lines, KARPAS-299 and SR-786, and two cutaneous T-cell lymphoma cell lines, HH and HuT-78 (Sézary syndrome), were used. Cell viability and apoptosis were studied after exposure to different increasing concentrations of the drug at 24, 48, and 72 hours by colorimetric MTT and annexin V/propidium iodide double-labeling assays, respectively. Amiloride significantly inhibited cell growth in the PTCL cell lines, depending on increasing drug concentration and time. Inhibition was statistically significant (P<0.05) from 0.15 mM for KARPAS-299, SR-786, and HH at 24, 48, and 72 hours and from 0.3 mM for HuT-78 at 48 and 72 hours. KARPAS-299 and SR-786 were the most sensitive cell lines to amiloride exposure, whereas HuT-78 cells were less sensitive, although with the highest concentration of amiloride, its cell viability decreased to levels below 50% and 40% at 48 and 72 hours, respectively. Induction of cell death by apoptosis was detected in all cell lines, with apoptosis levels up to 25% at 24 hours and more than 80% at 48 and 72 hours using intermediate doses of the drug. 1) Amiloride significantly decreased the cell viability of PTCL. 2) ALCL cell lines were the most sensitive to amiloride exposure. 3) The cytotoxic effect induced by amiloride in PTCL was mediated by apoptosis. Ongoing Objectives: To evaluate apoptosis as the possible mechanism of cell death induction through the analysis of caspases, synergism with drugs commonly used in the treatment of PTCL, and changes in gene and protein expression induced by amiloride. Regional Health Management of Castilla y León (GRS 2142/A/2020);); Carlos III Health Institute European Union (PI19/00674); AECC (PROYE20047GUTI).

IL-2/IL-2R signaling and IL-2Rα-targeted therapy in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is aCD30-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 anovel therapeutic alternative for ALCL patients.

1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42) inhibits cell proliferation and induces apoptosis via inhibiting ALK and its downstream pathways in Karpas299 cells

Anaplastic lymphoma kinase (ALK) belongs to the family of receptor tyrosine kinases. Recently, the incidence of anaplastic large cell lymphoma (ALCL) with ALK rearrangement has raised considerably. The application of ALK-targeted inhibitors such as ceritinib provides an effective therapy for the treatment of ALK-positive cancers. However, with the prolongation of treatment time, the emergence of resistance is inevitable. We found that 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42), a novel ceritinib derivative, could inhibit the proliferation of ALK-positive ALCL cells, induce the apoptosis of Karpas299 cells through the mitochondrial pathway in a caspase-dependent manner. In addition, ZX-42 could suppress ALK and downstream pathways including PI3K/Akt, Erk and JAK3/STAT3 and reduce the nuclear translocation of NFκB by inhibiting TRAF2/IKK/IκB pathway. Taken together, our findings indicate that ZX-42 shows more effective activity than ceritinib against ALK-positive ALCL. We hope this study can provide a direction for the structural modification of ceritinib and lay the foundation for the further development of clinical research in ALK-positive ALCL.

PB2171: THE NOVEL CSN5/JAB1 INHIBITOR CSN5I-3 SUPPRESSES CELL GROWTH AND UPREGULATES GENE EXPRESSION OF TYPE-I INTERFERONS (IFN) IN ANAPLASTIC LARGE CELL LYMPHOMA (ALCL)

Background: The CSN5/Jab1 (cJun activation domain-binding protein 1), initially discovered as a cJun coactivator, represents the fifth component of an evolutionary highly conserved 8 subunit protein complex, named COP9 signalosome (CSN5). Accumulating evidence suggests that the CSN5/Jab1 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via CDK inhibitors. We have previously shown that CSN5/Jab1 protein is overexpressed in T-cell lymphomas with the highest frequency of positive tumors being observed in both ALK+ and ALK- anaplastic large cell lymphomas (ALCL). Targeting CSN5/Jab1 activity in cancer is now possible since a novel inhibitor, namely CSN5i-3 (Novartis), has been developed for clinical use. The potential role of CSN5/Jab1 in modulation of anti-tumor immune responses in cancer, and particularly in ALCL, is unknown to date. Aims: To investigate the effects of the selective CSN5i-3 inhibitor in cell growth and type-I interferon - associated anti-tumor immune responses in ALCL. Methods: The ALCL in vitro system included 5 cell lines (3 ALK+ and 2 ALK- ALCL cell lines). Cell growth and viability were assessed by trypan blue and MTT assays. The effects of the CSN5i-3 on G1-S cell cycle progression were assessed by BrdU incorporation assay and on apoptosis by Annexin V/AAD staining and flow cytometry. Expression of various cell cycle regulating proteins at baseline and experimental conditions were analysed by Westem blot analysis. Gene expression of type I interferons, including interferonbeta (IFN-β), CXCL10 and interferon gamma (IFN-y), as well as, STING and a control gene (GAPDH) at the RNA level, were analysed with qRT-PCR. The ALCL cell lines were treated with the specific CSN5i-3 inhibitor at different concentrations. Silencing of the CSN5/Jab1 gene was performed using transient transfection with specific siRNA construct using the Amaxa Nucleofector System (Lonza). Results: Treatment of ALCL cell lines with increasing concentrations of CSN5i-3 resulted in decreased protein levels of CSN5/Jab1, which was associated with significantly decreased cell growth but slight increase in apoptosis (10-15%). The effects on cell growth linked to downregulation of cell cycle progression were associated with upregulation of the CDK inhibitors p21, p27 and p57. Similarly, CSN5/Jab1 gene silencing resulted in decreased cell growth associated with increased expression of similar cell cycle inhibiting proteins. In addition, inhibition of CSN5/Jab1 activity by CSN5i-3 resulted in increased IFN-β and IFN-y mRNA levels in ALK+ ALCL cells, and CXCL10, IFN-y and STING mRNA levels in ALK- ALCL cells. Similarly, knocking down CSN5/Jab1 gene by transient transfection with siRNA led to upregulation of type-I interferons, particularly IFN-β and CXCL10 in ALCL, suggesting that the effects of CSN5i-3 can be attributed to specific inhibition of CSN5/Jab1 activity. Summary/Conclusion: The novel inhibitor CSN5i-3 sufficiently downregulates expression of CSN5/Jab1 oncogene, inhibits cell cycle progression and contributes to type-I interferon - associated anti-tumor immune response in ALK+ and ALK- ALCL. These preclinical findings suggest a potential role of CSN5i-3 in molecular therapeutics and immunotherapy in T-cell lymphomas.

CD147 a direct target of miR-146a supports energy metabolism and promotes tumor growth in ALK+ ALCL

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 [18F]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.

NIPA (Nuclear Interaction Partner of ALK) Is Crucial for Effective NPM-ALK Mediated Lymphomagenesis.

The NPM-ALK fusion kinase is expressed in 60% of systemic anaplastic large-cell lymphomas (ALCL). A Nuclear Interaction Partner of ALK (NIPA) was identified as a binding partner of NPM-ALK. To identify the precise role of NIPA for NPM-ALK-driven lymphomagenesis, we investigated various NPM-ALK+ cell lines and mouse models. Nipa deletion in primary mouse embryonic fibroblasts resulted in reduced transformation ability and colony formation upon NPM-ALK expression. Downregulating NIPA in murine NPM-ALK+ Ba/F3 and human ALCL cells decreased their proliferation ability and demonstrated synergistic effects of ALK inhibition and NIPA knockdown. Comprehensive in vivo analyses using short- and long-latency transplantation mouse models with NPM-ALK+ bone marrow (BM) revealed that Nipa deletion inhibited NPM-ALK-induced tumorigenesis with prolonged survival and reduced spleen colonies. To avoid off-target effects, we combined Nipa deletion and NPM-ALK expression exclusively in T cells using a lineage-restricted murine ALCL-like model resembling human disease: control mice died from neoplastic T-cell infiltration, whereas mice transplanted with Lck-CreTG/wtNipaflox/flox NPM-ALK+ BM showed significantly prolonged survival. Immunophenotypic analyses indicated a characteristic ALCL-like phenotype in all recipients but revealed fewer “stem-cell-like” features of Nipa-deficient lymphomas compared to controls. Our results identify NIPA as a crucial player in effective NPM-ALK-driven ALCL-like disease in clinically relevant murine and cell-based models.

Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma

Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.