Anaplastic Large Cell Lymphoma Cells Research Articles

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.

The NPM-ALK Oncogenic Kinase Suppresses CGAS-Sting-Associated Anti-Tumor Immune Responses through STAT3 Activation in Anaplastic Large Cell Lymphoma (ALCL)

Background: ALK+ anaplastic large cell lymphoma (ALCL) is a distinct T-cell non-Hodgkin lymphoma type that frequently carries the t(2;5) resulting in overexpression and activation of NPM-ALK chimeric kinase, which activates multiple oncogenic pathways including JAK-STAT3 pathway. The presence of cytosolic DNA of either exogenous or endogenous origin activates the cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, which activates the adaptor protein STING. The latter then activates the TBK1 and IKK kinases, which activate through phosphorylation the transcription factors IRF3 and NF-κB, respectively. IRF3 and NF-κB induce expression of interferons (e.g. IFN-β) and cytokines leading to activation of innate immune responses. The potential role of NPM-ALK oncogenic kinase in cGAS-STING-related anti-tumor immune responses in ALK+ ALCL is unknown to date. Therefore, the present study aimed to investigate the biologic impact of NPM-ALK on cGAS-STING activation status and expression of relevant interferon genes in ALK+ ALCL.Methods: The in vitro system included 5 ALK+ (Karpas 299, SUPM2, DEL, SUDHL1, L82) and 2 ALK- (Mac-1, Mac-2a) ALCL cell lines, as well as Ba/F3 cells stably transfected with NPM-ALK (Ba/F3-NPM-ALK) or a control (Ba/F3-MIG) plasmid. Expression and activation (phosphorylation) of cGAS-STING pathway proteins at baseline and experimental conditions were analysed by RT-PCR and Western blot at the RNA and protein level, respectively. Inhibition of ALK and STAT3 activity was performed using Crizotinib and the selective XIII STAT3 inhibitor, respectively. Silencing of ALK gene was performed using transient transfection with ALK siRNA and the Amaxa Nucleofector Technology. A STING agonist and TBK inhibitor (Amlexanox) were also used alone or in combination with other agents. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of interferon beta (IFN-β), CXCL10, and interferon gamma (IFN-γ), as well as a control gene (GAPDH), with quantitative RT-PCR. The patient study group included 38 previously untreated patients with ALK+ ALCL. Immunohistochemical analysis for STING protein expression was performed using a monoclonal antibody (Cell Signaling) and standard protocols. An arbitrary 10% cutoff was used to define positivity.Results: STING gene was highly expressed at both the mRNA and protein level in ALK+ and ALK- ALCL cell lines, however, cGAS-STING pathway proteins were activated at a variable level among ALCL cell lines as shown in immunoblots. STING was highly expressed in 36 of 38 (95%) ALK+ ALCL tumors, highlighting its biologic significance in this lymphoma type. Inhibition of ALK activity by Crizotinib resulted in significant increase in IFN-β and CXCL10 gene expression linked to activation/phosphorylation of TBK1 indicating cGAS-STING pathway activation in ALK+ ALCL and Ba/F3-NPM-ALK cells. Silencing of ALK gene with specific ALK siRNA also resulted in a dramatic increase in the CXCL10 gene expression (mRNA level). Similarly, treatment of ALK+ ALCL cells with the XIII STAT3 inhibitor resulted in significantly increased IFN-β and CXCL10 gene expression associated with activation of the cGAS-STING pathway proteins in ALK+ ALCL cells but also in the ALK- ALCL cell line, Mac-1. Incubation of ALK+ ALCL cells with a STING agonist alone led to further activation of the cGAS-STING pathway in ALK+ ALCL cells.Conclusion: NPM-ALK suppresses STING-associated, anti-tumor immune responses in ALK+ ALCL, through STAT3 activation and regulation of gene expression of type-1 interferons (IFN-β, CXCL10). Thus, combined ALK inhibition (ALK inhibitors) and STING stimulation (STING agonists) may represent a novel investigational therapeutic strategy for these patients. DisclosuresNo relevant conflicts of interest to declare.

Aberrant Expression of and Cell Death Induction by Engagement of the MHC-II Chaperone CD74 in Anaplastic Large Cell Lymphoma (ALCL).

Simple SummaryAnaplastic large cell lymphoma (ALCL) is a lymphoid malignancy considered to be derived from T cells. Currently, two types of systemic ALCL are distinguished: anaplastic lymphoma kinase (ALK)-positive and ALK-negative ALCL. Although ALK+ and ALK− ALCL differ at the genomic and molecular levels, various key biological and molecular features are highly similar between both entities. We have developed the concept that both ALCL entities share a common principle of pathogenesis. In support of this concept, we here describe a common deregulation of CD74, which is usually not expressed in T cells, in ALCL. Ligation of CD74 induces cell death of ALCL cells in various conditions, and an anti-CD74-directed antibody-drug conjugate efficiently kills ALCL cell lines. Furthermore, we reveal expression of the proto-oncogene and known CD74 interaction partner MET in a fraction of ALCL cases. These data give insights into ALCL pathogenesis and might help to develop new treatment strategies for ALCL.In 50–60% of cases, systemic anaplastic large cell lymphoma (ALCL) is characterized by the t(2;5)(p23;q35) or one of its variants, considered to be causative for anaplastic lymphoma kinase (ALK)-positive (ALK+) ALCL. Key pathogenic events in ALK-negative (ALK−) ALCL are less well defined. We have previously shown that deregulation of oncogenic genes surrounding the chromosomal breakpoints on 2p and 5q is a unifying feature of both ALK+ and ALK− ALCL and predisposes for occurrence of t(2;5). Here, we report that the invariant chain of the MHC-II complex CD74 or li, which is encoded on 5q32, can act as signaling molecule, and whose expression in lymphoid cells is usually restricted to B cells, is aberrantly expressed in T cell-derived ALCL. Accordingly, ALCL shows an altered DNA methylation pattern of the CD74 locus compared to benign T cells. Functionally, CD74 ligation induces cell death of ALCL cells. Furthermore, CD74 engagement enhances the cytotoxic effects of conventional chemotherapeutics in ALCL cell lines, as well as the action of the ALK-inhibitor crizotinib in ALK+ ALCL or of CD95 death-receptor signaling in ALK− ALCL. Additionally, a subset of ALCL cases expresses the proto-oncogene MET, which can form signaling complexes together with CD74. Finally, we demonstrate that the CD74-targeting antibody-drug conjugate STRO-001 efficiently and specifically kills CD74-positive ALCL cell lines in vitro. Taken together, these findings enabled us to demonstrate aberrant CD74-expression in ALCL cells, which might serve as tool for the development of new treatment strategies for this lymphoma entity.

Super-enhancer-based identification of a BATF3/IL-2R\u2212module reveals vulnerabilities in anaplastic large cell lymphoma

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.

Pharmacological inhibitors of anaplastic lymphoma kinase (ALK) induce immunogenic cell death through on-target effects

Immunogenic cell death (ICD) is clinically relevant because cytotoxicants that kill malignant cells via ICD elicit anticancer immune responses that prolong the effects of chemotherapies beyond treatment discontinuation. ICD is characterized by a series of stereotyped changes that increase the immunogenicity of dying cells: exposure of calreticulin on the cell surface, release of ATP and high mobility group box 1 protein, as well as a type I interferon response. Here, we examined the possibility that inhibition of an oncogenic kinase, anaplastic lymphoma kinase (ALK), might trigger ICD in anaplastic large cell lymphoma (ALCL) in which ALK is activated due to a chromosomal translocation. Multiple lines of evidence plead in favor of specific ICD-inducing effects of crizotinib and ceritinib in ALK-dependent ALCL: (i) they induce ICD stigmata at pharmacologically relevant, low concentrations; (ii) can be mimicked in their ICD-inducing effects by ALK knockdown; (iii) lose their effects in the context of resistance-conferring ALK mutants; (iv) ICD-inducing effects are mimicked by inhibition of the signal transduction pathways operating downstream of ALK. When ceritinib-treated murine ALK-expressing ALCL cells were inoculated into the left flank of immunocompetent syngeneic mice, they induced an immune response that slowed down the growth of live ALCL cells implanted in the right flank. Although ceritinib induced a transient shrinkage of tumors in lymphoma-bearing mice, irrespective of their immunocompetence, relapses occurred more frequently in the context of immunodeficiency, reducing the effects of ceritinib on survival by approximately 50%. Complete cure only occurred in immunocompetent mice and conferred protection to rechallenge with the same ALK-expressing lymphoma but not with another unrelated lymphoma. Moreover, immunotherapy with PD-1 blockade tended to increase cure rates. Altogether, these results support the contention that specific ALK inhibition stimulates the immune system by inducing ICD in ALK-positive ALCL.

Abstract 1113: Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models

Abstract Neoplastic cells are characterized by genetic instability, leading to the random accumulation of mutations in different tumor subpopulations, which therefore respond differently to treatments. Given the average mutational rate of tumors, the probability to acquire resistance to a single drug usually exceeds the number of cells present at diagnosis, making drug resistance a fact in most cases. Statistically, the chance of a cell being resistant to two simultaneous treatments is much lower, suggesting that drug combinations may help prevent resistance. Anaplastic large cell lymphoma (ALCL) is an aggressive type of Non-Hodgkin Lymphoma, characterized by expression of the NPM/ALK oncogenic fusion tyrosine kinase. ALK+ ALCL patients resistant or relapsed (R/R) to front-line chemotherapy have a very poor prognosis. The therapeutic activity of the ALK inhibitor crizotinib has been shown in patients with R/R ALK+ ALCL. However, 30-40% of patients develop resistance to crizotinib monotherapy and relapse within short time, leaving no further therapeutic option than bone marrow transplant, when feasible. This highlights the need to identify new first-line therapies for high-risk patients.We explored the potential of upfront drug combinations to prevent the rise of resistant disease, in vitro and in vivo. Crizotinib was combined with CHOP chemotherapy, an epigenetic drug (decitabine), another targeted inhibitor (trametinib- MEK inhibitor), or a second-generation ALK inhibitor. ALK+ ALCL cells were kept in culture for ~100 days in the presence of different single or combined drugs. We found that in most cases, combined treatments were more effective than single agents in the long-term control of cells expansion. Upon further functional characterization using quantitative PCR and western blot, we found that combination treatments upregulated cell cycle inhibitors and pro-apoptotic genes as well as modulated survival factors to induce deeper inhibition of oncogenic signalling and higher rates of apoptosis. Crizotinib+decitabine combination treatment promoted downregulation of DNMT1 and increase of TGFB1, both of which indirectly affect cell cycle progression. In murine model, crizotinib+decitabine significantly delayed tumour growth, compared to single treatments. Our results indicate that combining a selective drug with a less specific anticancer agent, which allows unspecific killing of resistant clones that might potentially arise, would be beneficial.In conclusion, our data suggest that a polypharmacology approach combining ALK inhibition with other agents could be a valuable therapeutic option for ALK+ ALCL patients, to prevent relapses and improve outcomes. Citation Format: Geeta G. Sharma, Giulia Arosio, Matteo Villa, Mario Mauri, Marina Zappa, Vera Magistroni, Monica Ceccon, Sara Redaelli, Ilaria Crespiatico, Diletta Fontana, Cristina Mastini, Anna Garbin, Federica Lovisa, Lara Mussolin, Rocco Piazza, Carlo Gambacorrti-Passerini, Luca Mologni. Synergistic drug combinations prevent drug resistance in anaplastic large cell lymphoma preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1113.

NPM1-ALK Overexpression-Driven Toxicity in ALCL Is Partner Dependent and Mediated By STAT1 Antagonism of STAT3 Survival Signaling, Revealing a Novel Therapeutic Strategy

Activation of the tyrosine-kinase domain (TKD) of anaplastic lymphoma kinase (ALK) drives malignant growth in subsets of several cancers as part of oncogenic fusions resulting from chromosomal rearrangements. Clinically, 70% of anaplastic large-cell lymphoma (ALCL) and ~5% of non-small-cell lung cancer (NSCLC) are ALK+. ALK tyrosine-kinase inhibitors (TKIs) are approved for treatment of NSCLC and are under investigation in ALCL. Clinical resistance in lung cancer typically develops within 10-12 months and is due most commonly to activation of alternate pathways or second-site kinase-domain mutations. In contrast, in ALK + ALCL, we and others have shown that over-expression of the NPM1-ALK fusion kinase is the major mechanism of TKI resistance. While this permits cells to survive TKI treatment, it results in a toxic signaling overdose upon inhibitor withdrawal both in vitro and in vivo. Therefore, NPM1-ALK is both essential to the survival of ALK+ ALCL and able to induce apoptosis when activated above a certain threshold. We now report the mechanism underlying NPM1-ALK signaling overdose.We find that all generations of ALK TKIs drive NPM1-ALK overexpression as the most frequent resistance mechanism in ALCL cells, but increased ALK activity upon inhibitor withdrawal is universally toxic. ALK stimulates several oncogenic pathways, including JAK/STAT3, MEK/ERK, and PI3K/AKT, but we find none of these canonical downstream pathways is a key driver of overdose toxicity. Novel analysis of viability data using a normalized weighted area under the curve to quantify level of rescue showed that only ALK inhibitors can rescue from ALK overdose signaling. We also observed that NPM1-ALK over-expression has a significantly more detrimental effect on cell fitness than the lung cancer fusion EML4-ALK, suggesting that the toxicity seen upon overdose is fusion-partner dependent. For confirmation, we generated constitutively active mutants of the ALK TKD with no fusion partner that are potently transforming but do not impair cell fitness upon ALK overdose. Phosphoproteomics analysis of resistant sub-clones during overdose as compared to parental lines in vehicle show an overlap of over 1,700 targets driven by ALK signaling. Furthermore, comparison of the parental line in drug to the resistant line undergoing overdose, 178 genes group together in the overall death state. These analyses revealed 21 phospho targets uniquely upregulated during ALK overdose (p <0.05). Annotation of these genes align with RNA-seq data showing increased phosphorylation of proteins driving the same pathways.STAT1 was a prominent target further pursued as the driver of apoptosis during NPMI-ALK overdose. Normally activated by INF-γ, STAT1 antagonizes the transcriptional output of STAT3, the core survival pathway of ALCL. It previously was shown to be a direct phospho target of NPM1-ALK, which at steady state results in its proteasomal degradation permitting predominance of the STAT3 transcriptional program. Upon NPM-ALK overdose, however, we see rapid and dramatic upregulation of phosphorylated STAT1 (Y705), in both the cytoplasm and nucleus. RNA-Seq analysis at the gene level shows loss of STAT3 targets but up-regulation of the STAT1 program, suggesting that during NPM1-ALK overdose STAT1 activation antagonizes STAT3 survival output required for ALCL cell viability and driving them to apoptosis. IFN-γ, already known to be detrimental to ALK+ ALCL cells as a single agent, combined with ALK inhibitors therefore represents a novel treatment strategy able to synergistically deactivate the core survival pathway of ALK+ ALCL. DisclosuresNo relevant conflicts of interest to declare.

CDC37 As a Novel Target for the Treatment of NPM1-ALK Expressing Anaplastic Large Cell Lymphomas

Anaplastic large cell lymphoma (ALCL) is an aggressive CD30+ T-cell lymphoma that accounts for 2-8% and 10-15% of non-Hodgkin lymphomas in adults and children, respectively. The currently used standard therapy for anaplastic lymphoma kinase (ALK, a member of the insulin receptor superfamily) positive ALCL has limited effectiveness, resulting in a substantial percentage of cases with poor outcomes, either failing to enter remission or relapse within a few months after starting treatment. Thus, there is a clear unmet clinical need for developing novel, effective and safer therapeutic strategies for ALCL. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Approximately 50% of ALCL cases are positive for the NPM1-ALK fusion chimera generated by the t(2;5) chromosomal translocation. The oligomerization domain of NPM1 in the fusion protein NPM1-ALK mediates the ligand-independent dimerization of chimeric protein, which results in constitutive activation of the chimeric tyrosine kinase activity leading to downstream signaling pathways responsible for the oncogenicity. As NPM1-ALK is an established heat shock protein 90 (HSP90) client protein, we hypothesized that disruption of the interaction between master chaperone HSP90 and co-chaperone Cdc37 would be a viable druggable target. Celastrol is a natural compound extracted from the root bark of Chinese medicinal plant Tripterygium wilfordi. Anticancer properties of celastrol are attributed to its action on the interaction of HSP90 and its co-chaperone Cdc37. Hsp90 is an ATP-dependent master molecular chaperone. Cdc37 is a Hsp90 co-chaperone and disruption of Hsp90-Cdc37 interaction will result in the degradation of Hsp90 client proteins. Celastrol disrupts the interaction of Hsp90 and Cdc37 through inhibition of Hsp90 ATPase activity without blockage of ATP binding site as observed with Hsp90 inhibitors. We used NPM1-ALK endogenously expressing Karpas299, SUDHL-1 and ectopically expressing Ba/F3-FG-NPM1-ALK cell lines in our study. Treatment with celastrol (0.1-1 µM) significantly induced apoptosis dose dependently in ALCL cells. Apoptosis was associated with activated caspase 3 and poly (ADP-ribose) polymerase cleavage. We also observed upregulation of proapoptotic protein BAD and downregulation of antiapoptotic proteins survivin and MCL1. Celastrol treatment inhibited NPM1-ALK mediated signaling and downstream signaling effectors STAT3, AKT, and ERK1/2 by degradation of the NPM1-ALK fusion protein. Cell survival was evaluated by clonogenic survival assay and results were clearly indicative that the clonogenic survival is diminished in celastrol treated samples compared to controls. Our immunoprecipitation studies clearly demonstrated that celastrol treatment disrupted the interaction between Hsp90 and Cdc37 and induced degradation of the NPM1-ALK fusion protein. Altogether, targeting Cdc37 using celastrol is a novel therapeutic approach to induce apoptosis in ALCL cells expressing NPM1-ALK and warrants developing future therapeutic intervention strategies. DisclosuresGanguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau.

Single Agent and Synergistic Combinatorial Efficacy of First-in-Class Small Molecule Imipridone ONC201 in Hematological Malignancies In Vitro and In Vivo through Apoptosis Via the Integrated Stress Response Pathway

▪ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies.ONC201 was evaluated in 20 human leukemia cell lines (Figure 1): 13 acute myeloid leukemia (AML), 5 acute lymphoblastic leukemia (ALL) and 2 chronic myelogenous leukemia (CML) cell lines. We observed a dose-dependent decrease in cell viability in AML, ALL and CML cells (GI50 1-8 µM). ONC201 also decreased cell viability in 2 primary chronic lymphocytic leukemia (CLL) patient samples with 48-72h treatment. Dose- and time-dependent induction of apoptosis (caspase-3 cleavage), inhibition of Akt and Foxo3a phosphorylation was observed in AML cells treated with ONC201 for 24-48 hr. Sub-G1 analysis confirmed that ONC201 induced apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. ONC201-mediated apoptosis in ALL cells involved PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim was upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells, which are known to confer a poor clinical prognosis.Low micromolar concentrations of ONC201 also reduced multiple myeloma (MM) cell viability in a time- and dose-dependent manner. ONC201 reduced cell viability and induced apoptosis in dexamethasone-resistant MM.1R cells with efficacy comparable to dexamethasone-sensitive MM.1S cells (Figure 2). ONC201 caused activation of the integrated stress response (ISR) pathway, as observed with induction of ATF/CHOP in MM cells. XIAP is highly up-regulated in MM cells and sownregulation of IAP family proteins induces apoptosis. ONC201 treatment of MM cells resulted in a dose-dependent decrease in the anti-apoptotic proteins XIAP, cIAP1 and cIAP2.Next, ONC201 was evaluated in 33 human lymphoma cell lines: 3 anaplastic large cell lymphoma (ALCL), 7 Burkitt's lymphoma, 2 cutaneous T-cell lymphoma (CTCL), 8 diffuse large B-cell lymphoma (DLBCL), 1 MCL, 2 nodular sclerosis (NS) and 10 other lymphoma cell lines. ONC201 demonstrated low micromolar range efficacy (GI50 1-6 µM) across all lymphoma subtypes. Low micromolar concentrations of ONC201 reduced cell viability of MCL and DLBCL cell lines and primary cells in a time- and dose-dependent manner (Figure 3). ONC201 caused activation of the ISR pathway, as observed with induction of ATF4 in Pfeiffer DLBCL cells. In patient-derived MCL cells, ONC201 reduced cyclin D1 expression, a diagnostic feature of MCL.We then tested the efficacy of ONC201 in combination with approved therapies in hematological malignancies in vitro and in vivo. ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine confirmed by combination indices in AML cell lines CMK, THP-1, HL60 and ALL Kasumi-2 cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model exhibited superior tumor growth inhibition compared to either agent alone (Figure 4). ONC201 demonstrated synergistic reduction in cell viability in combination with 5-azacytidine that was confirmed by combination indices in AML cell lines HL-60, MOLM14 and MV411. ONC201 synergistically reduced cell viability in combination with dexamethasone or ixazomib in RPMI-8226 MM cells. ONC201 in combination with bortezomib synergistically reduced cell viability in KARPAS 299 ALCL cells and RPMI-8226 MM cells and improved apoptosis induction in UPN2 MCL cells. ONC201 combined with bortezomib was evaluated in a Burkitt's lymphoma xenograft model. Histological evaluation of tumor xenografts revealed a reduced tumor cell density at 3-4 weeks and enhanced CHOP induction at 48 hours with combination treatment compared to either agent alone. The combination treatment did not result in body weight loss.These results serve as a rationale for ongoing ONC201 single agent trials in relapsed/refractory acute leukemia (NCT02392572), non-Hodgkin's lymphoma (NCT02420795), MM (NCT02609230) and combination trial with dexamethasone in MM (NCT02863991). Our findings provide pharmacodynamic biomarkers and identify synergistic combinatorial regimens that are well tolerated and can be explored in the clinic. [Display omitted] DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc: Equity Ownership, Patents & Royalties.

MDMX/MDM4 is highly expressed and contributes to cell growth and survival in anaplastic large cell lymphoma

We hypothesized that murine double minute X (MDMX), a negative p53-regulator, may be involved in dysfunctional p53-signaling in anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-positive and ALK-negative, characterized frequently by non-mutated TP53 (wt-p53). By western blot analysis, MDMX was highly expressed in ALK + ALCL and expressed at variable levels in ALK- ALCL cell lines. By immunohistochemistry, high MDMX levels were observed more frequently in ALK + ALCL (36/46; 78%), compared with ALK- ALCL tumors (12/29; 41%) (p < .0018, Mann–Whitney-test). FISH analysis showed MDMX-amplification in 1 of 13 (8%) ALK- ALCL tumors, and low-level MDMX copy gains in 2 of 13 (15%) ALK- ALCL and 3 of 11 (27%) ALK + ALCL tumors. MDMX-pharmacologic inhibition or siRNA-mediated MDMX-silencing were associated with activated p53 signaling, growth inhibition and apoptotic cell death in wt-p53 ALCL cells, providing evidence that targeting MDMX may provide a new therapeutic approach for ALCL patients with wt-p53.

Increased Tenascin C, Osteopontin and HSP90 Levels in Plasmatic Small Extracellular Vesicles of Pediatric ALK-Positive Anaplastic Large Cell Lymphoma: New Prognostic Biomarkers?

Over the past 15 years, several biological and pathological characteristics proved their significance in pediatric anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL) prognostic stratification. However, the identification of new non-invasive disease biomarkers, relying on the most important disease mechanisms, is still necessary. In recent years, plasmatic circulating small extracellular vesicles (S-EVs) gathered great importance both as stable biomarker carriers and active players in tumorigenesis. In the present work, we performed a comprehensive study on the proteomic composition of plasmatic S-EVs of pediatric ALCL patients compared to healthy donors (HDs). By using a mass spectrometry-based proteomics approach, we identified 50 proteins significantly overrepresented in S-EVs of ALCL patients. Gene Ontology enrichment analysis disclosed cellular components and molecular functions connected with S-EV origin and vesicular trafficking, whereas cell adhesion, glycosaminoglycan metabolic process, extracellular matrix organization, collagen fibril organization and acute phase response were the most enriched biological processes. Of importance, consistently with the presence of nucleophosmin (NPM)-ALK fusion protein in ALCL cells, a topological enrichment analysis based on Reactome- and Kyoto Encyclopedia of Genes and Genomes (KEGG)-derived networks highlighted a dramatic increase in proteins of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in ALCL S-EVs, which included heat shock protein 90-kDa isoform alpha 1 (HSP90AA1), osteopontin (SPP1/OPN) and tenascin C (TNC). These results were validated by Western blotting analysis on a panel of ALCL and HD cases. Further research is warranted to better define the role of these S-EV proteins as diagnostic and, possibly, prognostic parameters at diagnosis and for ALCL disease monitoring.

B7-H3 Chimeric Antigen Receptor Redirected T Cells Target Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma.

Simple SummaryAlthough chemotherapy is associated with high relapse rates and numerous side effects, it is still used as the front line treatment of anaplastic large cell lymphoma (ALCL). Therefore, alternative treatment options for ALCL are needed. In this study, we show that B7-H3 is a novel and promising target in ALCLs, and demonstrate that B7-H3 directed chimeric antigen receptor (CAR) T cells have therapeutic potency in controlling ALCL tumor growth.Potent CAR-T therapies that target appropriate antigens can benefit the treatment of anaplastic lymphoma kinase-positive (ALK+) anaplastic large cell lymphoma (ALCL), which is the most common subtype of T cell lymphoma. In this study, we observed overexpression of B7-H3 in ALCL cell lines derived from clinical samples and differential expression of B7-H3 in an ALK-induced T cell transformation model. A B7-H3-redirected CAR based on scFv from mAb 376.96 was developed. B7-H3 CAR-T cells showed strong cytotoxicity and cytokine secretion against target ALCL cells (SUP-M2, SU-DHL-1, and Karpas 299) in vitro. Furthermore, the B7-H3 CAR-T cells exhibited proliferative capacity and a memory phenotype upon repeated antigen stimulation. We demonstrated that B7-H3 CAR-T cells could promptly eradicate ALCL in murine xenografts. Taken together, B7-H3 is a novel and promising target in ALCLs and B7-H3 CAR-T may be a viable treatment option for ALCL.

Nucleophosmin-anaplastic lymphoma kinase of anaplastic large-cell lymphoma recruits, activates, and uses pp60c-src to mediate its mitogenicity

Anaplastic large-cell lymphomas (ALCLs) are lymphomas of T or null phenotype often associated with a chromosomal translocation, t(2;5)(p23;q35). This translocation leads to the expression of a hybrid protein consisting of the N-terminal portion of nucleophosmin (NPM) and the intracellular domain of the anaplastic lymphoma kinase (ALK). NPM-ALK possesses a constitutive tyrosine kinase activity responsible for its oncogenic property through activation of downstream effectors such as phospholipase C gamma (PLC-gamma) and the type IA phosphoinositide 3-kinase. Here, we show that the Src-kinases, particularly pp60(c-src), associate with and are activated by NPM-ALK expression in various cells, and in cell lines established from patients with ALCL. The kinase activity and the tyrosine 418 of NPM-ALK are required for its association with Src-kinases. Y418F mutation of NPM-ALK impaired its association with Src-kinases and strongly reduced the proliferation rate of Ba/F3 cells. In agreement, Src-kinase inhibitors or pp60(c-src) siRNA significantly decreased the proliferation rate of NPM-ALK-positive ALCL cell lines. Moreover, using active or inactive forms of pp60(c-src) and NPM-ALK, we provide evidence that NPM-ALK is a potential substrate of pp60(c-src). Overall, our data place Src-kinases as new important downstream effectors of NPM-ALK and as attractive potential therapeutic targets for new ALCL treatment.

EBP2, a novel NPM-ALK-interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53.

The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found in anaplastic large-cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remains unclear. We herein demonstrated that NPM-ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM-ALK interacted with Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM-ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G0 /G1 -phase cell cycle arrest in Ba/F3 cells transformed by NPM-ALK and ALCL patient-derived Ki-JK cells, but not ALCL patient-derived SUDH-L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM-ALK and Ki-JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC-0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, an essential component of mTORC1. These results suggest that the knockdown of EBP2 triggered p53 activation through the Akt-mTORC1 pathway in NPM-ALK-positive cells. Collectively, the present results revealed the critical repressive mechanism of p53 activity by EBP2 and provide a novel therapeutic strategy for the treatment of ALCL.

A novel model of alternative NF-κB pathway activation in anaplastic large cell lymphoma.

Aberrant activation of NF-κB is the most striking oncogenic mechanism in B-cell lymphoma; however, its role in anaplastic large cell lymphomas (ALCL) has not been fully established and its activation mechanism(s) remain unclear. Using ALCL cell line models, we revealed the supporting roles for NFKB2 and the NIK pathway in some ALCL lines. To investigate the detailed activation mechanisms for this oncogenic pathway, we performed specifically designed alternative NF-κB reporter CRISPR screens followed by the RNA-seq analysis, which led us to identify STAT3 as the major mediator for NIK-dependent NF-κB activation in ALCL. Consistently, p-STAT3 level was correlated with NFKB2 nuclear accumulation in primary clinical samples. Mechanistically, we found that in NIK-positive ALK- ALCL cells, common JAK/STAT3 mutations promote transcriptional activity of STAT3 which directly regulates NFKB2 and CD30 expression. Endogenous expression of CD30 induces constitutive NF-κB activation through binding and degrading of TRAF3. In ALK+ ALCL, the CD30 pathway is blocked by the NPM-ALK oncoprotein, but STAT3 activity and resultant NFKB2 expression can still be induced by NPM-ALK, leading to minimal alternative NF-κB activation. Our data suggest combined NIK and JAK inhibitor therapy could benefit patients with NIK-positive ALK- ALCL carrying JAK/STAT3 somatic mutations.

Striking Association of Lymphoid Enhancing Factor (LEF1) Overexpression and DUSP22 rearrangements in Anaplastic Large Cell Lymphoma

Introduction: Anaplastic large cell lymphomas (ALCLs) are CD30-positive T-cell lymphomas classified into ALK-positive and ALK-negative. The ALK-negative ALCLs are a genetically heterogeneous group that includes DUSP22-rearranged, TP63-rearranged and triple-negative cases. The DUSP22-rearranged ALCLs characteristically grow in a diffuse sheet-like pattern and are composed of intermediate-sized monomorphous hallmark cells, admixed with smaller cells with prominent nuclear indentations and occasional central nuclear pseudo inclusions (King RL et al, Am J Surg Pathol. 2016). Further, DUSP22-rearranged ALCL is a molecularly distinct subset of ALCL with a unique immunogenic phenotype that portends a favorable prognosis (Luchtel RA et al. Blood 2018). Lymphoid enhancer binding factor (LEF1) is a crucial nuclear mediator of the Wnt/ β-catenin pathway and a transcription factor involved in T-cell maturation. However, limited data exists on the association of LEF1 in T-cell lymphomas, including ALCL. The aims of this study were to explore LEF1 expression by immunohistochemistry in different subsets of ALCLs and to analyze the association of Wnt/ β-catenin pathway in this cohort. Methods: After approval by the institutional review board, we screened pathology reports from January 1, 1995 to December 31, 2019 and identified patients with a diagnosis of ALCL using our institutional database. Details on fluorescence in situ hybridization studies in ALK-negative cases were documented. Immunostaining for LEF1 and β catenin was performed on all cases where formalin fixed paraffin embedded tissue was available. The presence or absence of β catenin nuclear staining was recorded. LEF1 nuclear staining in the neoplastic ALCL cells was graded as negative (0), weak (1+), intermediate (2+) or strong (3+) and the percentage of LEF1-positive neoplastic cells was detailed (Figure 1). LEF1 scoring was performed by manual counting of the stained nuclei out of all tumor nuclei by two pathologists (A.R. and M.S.) and the grading was reviewed by another pathologist (P.J.K.). Results: We evaluated 45 ALCL cases, including 41 (91.1%) ALK-negative and 4 (8.9%) ALK-positive ALCLs. Among the ALK-negative ALCLs, there were16 DUSP22-rearranged, and 25 non-DUSP22-rearanged (7 TP63-rearranged and 18 triple-negative) cases. Fifteen (94%) of 16 DUSP22-rearranged ALCLs showed strong expression of LEF1 in &amp;gt;75% tumor cells (Figure 2). In contrast, this strong and uniform LEF1 overexpression was only detected in 1 of 29 (3%) of the remaining ALCL cases (P &amp;lt;.0001) (Table 1). We previously characterized a distinctive molecular signature in ALK-negative ALCLs with DUSP22-rearrangements using gene expression microarray analysis on frozen ALCL tissue samples (Luchtel RA et al. Blood 2018). On examining LEF1 gene expression in this previously published dataset, DUSP22 -rearranged cases had significantly higher LEF1 expression than any other genetic subtype (triple-negative, TP63-rearranged, or ALK-positive). Overall, mean LEF1 expression was 6.3-fold higher in ALCLs with DUSP22 rearrangements than in ALCLs without DUSP22-rearrangements (P=0.0001). LEF1 is a coactivator of the Wnt/β-catenin pathway; therefore, it is reasonable to hypothesize the overexpression of LEF1 may promote Wnt/β-catenin signaling. However, all LEF1-positive cases were negative for β-catenin by immunohistochemistry, suggesting that LEF1 overexpression is independent of the Wnt/β-catenin pathway signaling in these cases. Conclusions: There is a striking association between high LEF1 expression and DUSP22-rearrangements in ALCLs. The strong and uniform LEF1 expression pattern has a high positive predictive value (94%) and high negative predictive value (96%) for DUSP22- rearrangement in ALK-negative ALCLs (Table 2). The combination of characteristic morphologic and molecular features of DUSP22-rearranged cases with the unique LEF1 expression further emphasizes that DUSP22-rearranged ALCL represents a distinct clinicopathologic subset of ALCL. Disclosures No relevant conflicts of interest to declare.

Abstract PO-50: Phophatases modulate resistance to ALK inhibitors in anaplastic large-cell lymphoma

Abstract Introduction: Anaplastic large-cell lymphomas (ALCL) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. The ALK fusions activate several signaling pathways, promoting cell growth, migration, and survival. Chemotherapy is the standard treatment for ALCL patients, but about 30% of patients relapse. Targeting ALK using tyrosine kinase inhibitors (TKIs) has shown promising results, and the FDA recently granted breakthrough therapy designation to crizotinib for use in patients with relapsed/refractory ALK+ ALCL. However, resistance to crizotinib develops in ALK+ ALCL patients secondary to ALK mutations or unknown mechanisms. In this study, we aimed at elucidating unknown bypass mechanisms of crizotinib resistance in ALK+ ALCL. Methods: We used Genome-wide CRISPR-Cas9 Knockout Screening (GeCKO.v2) to identify candidate genes that contribute to resistance to crizotinib. Four different ALCL cell lines were infected with Lenti-GeCKO libraries. After treatment with crizotinib for 14 days to select for resistant cells, next-generation sequencing was performed on crizotinib-resistant cells to identify candidate resistance genes. Top candidates were selected for validation assays and further analyses. Results: Genomic loss-of-function screens identified two phosphatases, PTPN1 and PTPN2, in all ALCL cell lines as consistent top hits driving resistance to crizotinib. Functional validation of these candidate genes showed that single loss of either PTPN1 or PTPN2 generates immediate resistance to crizotinib and other ALK TKIs such as alectinib and lorlatinib. Consistently, RNA-seq in patients who developed resistance to crizotinib showed downregulation of PTPN1 or PTPN2 expression. By multiple assays, we demonstrate that PTPN1 and PTPN2 are phosphatases that de-phosphorylate ALK, thereby regulating its overall phosphorylation levels and activity. In addition, we found that PTPN1, but not PTPN2, is also a phosphatase of SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating the MAPK and JAK/STAT pathways. A treatment that combined crizotinib and a recently developed SHP2 inhibitor (SHP099) completely blocked the sustained MAPK activation and reverted crizotinib resistance in vitro and in vivo. Conclusions: We discovered that PTPN1 and PTPN2 are ALK phosphatases that control sensitivity to ALK TKIs in ALCL. Combined inhibition of SHP2 is a potential therapeutic approach to overcome resistance to ALK TKIs in ALCL. Citation Format: Elif Atabay, Qi Wang, Taek-Chin Cheong, Nina Prokoph, Chiara Ambrogio, Ines Mota, Achille Pich, Enrico Patrucco, Claudia Voena, Roberto Chiarle. Phophatases modulate resistance to ALK inhibitors in anaplastic large-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-50.

The NKL-code for innate lymphoid cells reveals deregulated expression of NKL homeobox genes HHEX and HLX in anaplastic large cell lymphoma (ALCL).

NKL homeobox genes encode developmental transcription factors and display an NKL-code according to their physiological expression pattern in hematopoiesis. Here, we analyzed public transcriptome data from primary innate lymphoid cells (ILCs) for NKL homeobox gene activities and found that ILC3 expressed exclusively HHEX while in ILC1 and ILC2 these genes were silenced. Deregulation of the NKL-code promotes hematopoietic malignancies, including anaplastic large cell lymphoma (ALCL) which reportedly may derive from ILC3. Accordingly, we analyzed NKL homeobox gene activities in ALCL cell lines and investigated their role in this malignancy. Transcriptome analyses demonstrated low expression levels of HHEX but powerfully activated HLX. Forced expression of HHEX in ALCL cell lines induced genes involved in apoptosis and ILC3 differentiation, indicating tumor suppressor activity. ALCL associated NPM1-ALK and JAK-STAT3-signalling drove enhanced expression of HLX while discounting HHEX. Genomic profiling revealed copy number gains at the loci of HLX and STAT3 in addition to genes encoding both STAT3 regulators (AURKA, BCL3, JAK3, KPNB1, NAMPT, NFAT5, PIM3, ROCK1, SIX1, TPX2, WWOX) and targets (BATF3, IRF4, miR135b, miR21, RORC). Transcriptome data of ALCL cell lines showed absence of STAT3 mutations while MGA was mutated and downregulated, encoding a novel potential STAT3 repressor. Furthermore, enhanced IL17F-signalling activated HLX while TGFbeta-signalling inhibited HHEX expression. Taken together, our data extend the scope of the NKL-code for ILCs and spotlight aberrant expression of NKL homeobox gene HLX in ALCL. HLX represents a direct target of ALCL hallmark factor STAT3 and deregulates cell survival and differentiation in this malignancy.

Abstract 4715: Characterization of potential co-drivers of pathogenesis in ALK positive ALCL

Abstract ALK positive Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma characterized by expression of the NPM/ALK fusion tyrosine kinase essential for neoplastic cells growth. When resistant or relapsed to front-line chemotherapy, ALK+ ALCL prognosis is very poor. Although ALK+ ALCL cells are mainly driven by the constitutively active NPM/ALK kinase, presence of additional mutations might affect the disease course and response to treatment, leading to different outcomes in different patients. We employed whole exome sequencing to characterize the co-mutational burden in ALK+ ALCL samples at diagnosis. Diagnosis biopsy and matched healthy control DNA samples from 24 ALK+ ALCL patients were sequenced. Somatic variants present at high frequency within the tumor (at least 25% variant allele frequency), which can be viewed as potential co-drivers, were annotated; the identified mutations were weighed using in silico tools to predict their oncogenic potential and their effects on protein function. All identified variants were confirmed by Sanger sequencing. Gene ontology and pathway analyses identified cell adhesion and actin cytoskeleton among the most enriched biological functions. Using the above-mentioned criteria, we found recurrent somatic variants in 7 genes including PPP1R9A, RUNX1T1, FAT4 and RBBP8. Here we report the initial characterization of the role of FAT4 in ALCL cells. FAT4 is a member of the cadherin superfamily with tumor suppressor functions. In our cohort, 3/24 patients (13%) were found to carry FAT4 mutations, including nonsense mutations and missense changes predicted to be deleterious, suggesting a loss of function effect. To characterize the biological consequences of these mutations, we generated FAT4 knockdown ALCL cell lines. FAT4-silenced cells grew faster and showed increased migratory properties compared to parental cells. They also differed from parental cells in terms of morphology, having bigger size and modulated F-actin rearrangement. Tumor suppressing activity of FAT4 has been linked to Wnt/β-catenin pathway; we found that FAT4-silenced cells had decreased levels of serine/threonine phosphorylated β-catenin and higher nuclear localization, which insinuates activation of Wnt/β-catenin signalling and henceforth, increased cellular proliferation. To explore therapeutic implications of FAT4 mutations, we analyzed the sensitivity of FAT4 silenced cells to chemotherapeutic agents. FAT4 knockdown cells were less sensitive to a CHOP combination treatment as compared to parental cells. No difference was seen in terms of sensitivity to crizotinib. Finally, we investigated the effects of RUNX1T1 somatic variants found in our study. Preliminary luciferase assay results showed that the variants significantly increase the transcriptional repression ability of RUNX1T1. Altogether, our data provides information about genes other than NPM-ALK that might play a role in the pathogenesis of ALCL. Citation Format: Luca Mologni, Geeta G. Sharma, Matteo Villa, Mario Mauri, Giulia Arosio, Cosimo Lobello, Hugo Larose, Alessandra Pirola, Silvia Bombelli, Nicoletta Cordani, Luca Massimino, Šárka Pospíšilová, Suzanne D. Turner, Giorgio Inghirami, Fabio Pagni, Rocco Piazza, Roberto Perego, Roberto Chiarle, Carlo Gambacorti-Passerini. Characterization of potential co-drivers of pathogenesis in ALK positive ALCL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4715.

Aptamer-Equipped Protamine Nanomedicine for Precision Lymphoma Therapy.

Anaplastic large cell lymphoma (ALCL) is the most common T-cell lymphoma in children. ALCL cells characteristically express surface CD30 molecules and carry the pathogenic ALK oncogene, both of which are diagnostic biomarkers and are also potential therapeutic targets. For precision therapy, we report herein a protamine nanomedicine incorporated with oligonucleotide aptamers to selectively target lymphoma cells, a dsDNA/drug payload to efficiently kill targeted cells, and an siRNA to specifically silence ALK oncogenes. The aptamer-equipped protamine nanomedicine was simply fabricated through a non-covalent charge-force reaction. The products had uniform structure morphology under an electron microscope and a peak diameter of 103 nm by dynamic light scattering measurement. Additionally, flow cytometry analysis demonstrated that under CD30 aptamer guidance, the protamine nanomedicine specifically bound to lymphoma cells, but did not react to off-target cells in control experiments. Moreover, specific cell targeting and intracellular delivery of the nanomedicine were also validated by electron and confocal microscopy. Finally, functional studies demonstrated that, through combined cell-selective chemotherapy using a drug payload and oncogene-specific gene therapy using an siRNA, the protamine nanomedicine effectively killed lymphoma cells with little toxicity to off-target cells, indicating its potential for precision therapy.