Anaplastic Large Cell Lymphoma Tumors Research Articles

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.

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

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.

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.

Phase I Study of Yttrium-90 Labeled ANTI-CD25 (aTac) Monoclonal Antibody PLUS BEAM for Autologous Hematopoietic Cell Transplantation (AHCT) in Patients with Mature T-Cell NON-Hodgkin Lymphoma, the “a-TAC-BEAM Regimen”

Background: Peripheral T cell lymphomas (PTCL) have a poor prognosis with current treatment regimens. High-dose chemotherapy followed by autologous stem cell transplant (ASCT) has been used as a consolidation strategy in remission states (CR1 or above) endorsed by the NCCN guidelines in appropriate patients. 5-year DFS is reported at 70% for alk -ve anaplastic large cell lymphoma (ALCL) and 30-40% for most other histologies (D'Amore et al, 2012, JCO). It is also performed in the relapsed settings if no previous ASCT performed and allogeneic transplant is not an option. CD25 is a targetable protein expressed differentially in PTCL and antibody based anti-CD25 therapies are efficacious in PTCL i.e denileukin diftitox (Foss et al Blood 2006, Dang et al, BJH 2006) , monoclonal antibody dacluzimab (Waldman et al 1995 Blood). Yttrium-90 (90Y) labeled chimeric antiCD25 antibody basiliximab emits beta particles and has been shown to inhibit the growth of human ALCL tumors and increase survival in SUDHL-1 xenograft mice (Zhang et al 2009 Cancer Biother Radiopharm). Previous investigations at COH by Raubitschek, Colcher et al established a safe does of Yttrium-90 (90Y) labeled basiliximab at 0.4mCi/kg in combination with BEAM. This is a phase 1 clinical trial of a novel conditioning regimen that includes the use of Yttrium-90 (90Y) labeled basiliximab with BEAM chemotherapy for PTCL patients eligible for ASCT. The trial utilizes a modified version of the rolling 6 design (Skolnik et al) to test 3 dose levels of Yttrium-90 (90Y) Basiliximab i.e 0.4mCi/kg, 0.5miC/kg and 0.6mCi/kg with the primary objective of evaluating the safety and tolerability of this combination and to establish the MTD. Secondary objectives include estimating incidence of relapse, OS, PFS, NRM at day 100, 1 year and 2 years post-transplant. Patients and Methods: Dose limiting toxicity (DLT) is defined according to the Bearman and CTCAE 4.03 scales, the latter for hematologic toxicity. The study/treatment schema is shown in Figure 1. Results: From 07/29/2015 to 06/10/2020, 20 patients underwent ASCT on this trial; n=4 at 0.4mCi/kg n=4 at 0.5mCi/kg and n=12 at 0.6mCi/kg. Median age at ASCT was 51 years (range: 18-76), and histologies included; PTCL-nos (n=10); alk-ve ALCL (n=5); angioimmunoblastic T-cell lymphoma (n=3); and intestinal T-cell lymphoma (n=2). Disease status at ASCT were CR1 in18, CR2 in 2 patients. Median number of prior therapies was 1 (range: 1-4). At a median follow-up of 17.1 months (range: 0.9-26.2), 12 patients remain in remission, 8 have relapsed out of which 5 have died of progressive lymphoma. OS was 100% (95% CI: N/A) at 100-days, and 83% (95% CI: 57-94) at 1 year. Non-relapse Mortality was 0% at both 100-days and 1-year. All patients successfully engrafted with the median days to ANC &amp;gt;= 500/ul was 10 (range: 10 - 21), and days to PLT &amp;gt;= 20,000/ul: 13 (12 - 92). Overall, no dose limiting toxicities were experienced. The most common/highest grade toxicity experienced (per Bearman Scale) was grade 2 stomatitis, which was seen in 3 patients at 0.4mCi/kg; 4 patients at 0.5 mCi/kg, and 7 at0.6mCi/kg. The only other toxicities seen were grade 2 GI in 2 patients at 0.4mCi/kg, and grade 2 bladder in one patient at 0.6mCi/kg dose.. Toxicities &amp;gt;grade 2 were not seen. Conclusion: aTac- BEAM appears to be safe as an ASCT conditioning regimen for PTCL with no increased toxicity as compared to the historical toxicities seen with BEAM alone in this patient population (D'Amore 2012 J of Clin Onc). The dose level 0.6mCi/kg will likely be the recommended phase II dose. An expanded phase is planned to evaluate the efficacy of this regimen followed by a randomized trial of BEAM alone plus a combination of aTac- BEAM. Figure 1 Disclosures Zain: Mundi Pharma: Research Funding; Seattle Genetics: Research Funding; Kyowa Kirin: Research Funding. Herrera:Gilead Sciences: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Immune Design: Research Funding; AstraZeneca: Research Funding; Karyopharm: Consultancy; Pharmacyclics: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Other: Travel, Accomodations, Expenses, Research Funding; Merck: Consultancy, Research Funding. Salhotra:Kadmon: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Nakamura:NapaJen Pharma: Consultancy; Celgene: Other: Support on seminar; Magenta Therapeutics: Other: Advisory board meeting; Viracor: Consultancy; Merck: Other: advisory board meeting; Alexion: Other: Support on a meeting presentation; Kyowa-Kirin: Other: Support on a meeting presentation; Kadmon Corporation: Other: Advisory board meeting. OffLabel Disclosure: Yittrium labelled Basiliximab

GSK1838705a, an IGF-1R/ALK Inhibitor, Overcomes Resistance to Crizotinib in ALK-Positive ALCL

Anaplastic large cell lymphoma (ALCL) is a type of CD30-expressing non-Hodgkin's lymphoma (NHL), which accounts for 2% to 3% of adult non-Hodgkin's lymphoma,accounting for 15% to 30% of children with large cell lymphoma. Anaplastic lymphoma kinase (ALK) positive ALCL is highly invasive, and currently it is generally based on CHOP combined with chemotherapy. The proportion of patients with complete relief of symptoms is as high as 90%, but the proportion of recurrence is also as high as 40%. Crizotinib is the first generation of ALK inhibitors that have been approved for the treatment of ALK+ ALCL. Unfortunately, most patients treated with crizotinib relapse after a significant initial response. The median progression-free survival of clinical trials was 10.5 months. Various mutations in the ALK kinase domain and amplification of the ALK gene copy number, activation of the alternative pathway, and tumor heterogeneity are major causes of crizotinib resistance. Studies have shown that IGF-1R interacts with NPM-ALK to promote ALK+ALCL transformation, proliferation and migration. GSK is a small molecule kinase inhibitor that inhibits both IGF-IR and ALK. Therefore, GSK with simultaneous inhibition of the bidirectional potential of IGF-IR and ALK has a promising prospect in the targeted therapy of NPM-ALK+ALCL. This study explored the inhibitory effects of GSK on NPM-ALK+ALCL and crizotinib-resistant NPM-ALK+ALCL by in vivo and in vitro experiments. In vitro experiments: The sensitivity of ALCL cell line to GSK1838705a was detected by CCK8 and flow cytometry. The expression of phosphorylation of IGF-1R and NPM-ALK signaling pathway in Karpas299 and SR786 cell lines stimulated by GSK was detected by WB method. In order to study the crizotinib resistance mutation, we established ALK+ALCL crizotinib-resistant cell lines Karpas299-R and SR786-R, and identified the resistance of Karpas299-R and SR786-R cell lines by CCK8 and flow cytometry. The drug-resistant and non-resistant strains were stimulated with gradient concentrations of crizotinib and gradient GSK, and the IC50 of the two were compared by CCK8. The WB method was used to compare the phosphorylation levels of downstream signaling pathways in drug-resistant and non-resistant strains. In vivo experiment: The ALK+ALCL and resistant-ALK+ALCL mouse model was established, and three groups of mice treated with control, GSK single drug 30 mg/kg, GSK single drug 60 mg/kg, were established. The tumor volume and body weight of the four groups were compared. Immunohistochemistry was used to compare the expression levels of key signaling molecules and apoptotic proteins in each group. SPSS statistical software draws survival curves. As the concentration of GSK gradually increases, the survival rate of ALCL cells gradually decreases. The expression of pIGF-1R, pNPM-ALK, pSTAT3, pAKT, casepase3 and other molecules decreased in the downstream signaling pathway, and the expression level of cleaved-casepase3 increased.In the crizotinib-resistant cell line, with the increase of the concentration of GSK, the apoptosis rate of the cells increased and the phosphorylation level of the downstream molecules gradually decreased. Tumor volume of three groups of mouse models: control&gt;GSK single drug 30 mg/kg&gt;GSK single drug 60 mg/kg. Immunohistochemistry results showed that the expression level of key signaling molecules in GSK-treated CHOP-treated mice decreased, and the expression level of apoptotic proteins increased. In this research, we explored the effects of GSK1838705A on proliferation, apoptosis, and clonogenesis of ALCL cell lines. Subsequently, we established a crizotinib-resistant cell line and noticed that GSK1838705A can effectively reduce the viability of resistant ALCL cells and significantly restrain the transmission of downstream survival signaling pathways induced by IGF1R/IR phosphorylation. Besides, we discovered that GSK1838705A inhibited the development of both crizotinib-sensitive and crizotinib-resistant ALCL tumors in the ALCL mouse model established by subcutaneous tumorigenesis. Based on the results of previous clinical trials, we put forward to use GSK1838705A as an alternative treatment strategy to overcome crizotinib-resistant ALCL. Disclosures No relevant conflicts of interest to declare.

Phase I Study of Yttrium-90 Labeled ANTI-CD25 (aTac) Monoclonal Antibody PLUS BEAM for Autologous Hematopoietic CELL Transplantation (AHCT) in Patients with Mature T-CELL NON-Hodgkin Lymphoma, the “a-TAC-BEAM Regimen”

Background: Peripheral T cell lymphomas (PTCL) have a poor prognosis with current treatment regimens. High-dose chemotherapy followed by autologous stem cell transplant (ASCT) has been used as a consolidation strategy in remission states (CR1 or above) endorsed by the NCCN guidelines in appropriate patients. 5 year DFS is reported at 70% for alk -ve anaplastic large cell lymphoma (ALCL) and 30-40% for most other histologies (D'Amore et al, 2012, JCO). It is also performed in the relapsed settings if no previous ASCT performed and allogeneic transplant is not an option. CD25 is a targetable protein expressed differentially in PTCL and antibody based anti-CD25 therapies are efficacious in PTCL i.e denileukin diftitox (Foss et al Blood 2006, Dang et al , BJH 2006) , monoclonal antibody dacluzimab (Waldman et al 1995 Blood). Yttrium-90 (90Y) labeled chimeric antiCD25 antibody basiliximab emits beta particles and has been shown to inhibit the growth of human ALCL tumors and increase survival in SUDHL-1 xenograft mice (Zhang et al 2009 Cancer Biother Radiopharm). Previous investigations at COH by Raubitschek, Colcher et al established a safe does of Yttrium-90 (90Y) labeled basiliximab at 0.4mCi/kg in combination with BEAM. This is a phase 1 clinical trial of a novel conditioning regimen that includes the use of Yttrium-90 (90Y) labeled basiliximab with BEAM chemotherapy for PTCL patients eligible for ASCT. The trial utilizes a modified version of the rolling 6 design (Skolnik et al) to test 3 dose levels of Yttrium-90 (90Y) Basiliximab i.e 0.4mCi/kg, 0.5miC/kg and 0.6mCi/kg with the primary objective of evaluating the safety and tolerability of this combination and to establish the MTD. Secondary objectives include incidence of relapse, OS, PFS, NRM at day 100, 1 year and 2 years post-transplant.Patients and Methods: Dose limiting toxicity (DLT) is defined according to the Bearman and CTCAE 4.03 scales, the latter for hematologic toxicity. The study/treatment schema is shown in Figure 1.Results: From 07/29/2015 to 05/29/2018, 14 patients underwent ASCT on this trial; n=4 at 0.4mCi/kg n=4 at 0.5mCi/kg and n=6 at 0.6mCi/kg. Median age at ASCT was 51 years (range: 18-76), and histologies included; PTCL-nos (n=7); alk-ve ALCL (n=3); angioimmunoblastic T-cell lymphoma (n=2); and intestinal T-cell lymphoma (n=2). Disease status at ASCT were CR1 in12, CR2 in 2 patients. Median number of prior therapies was 1 (range: 1-2). At a median follow-up of 14.4 months (range: 0.9-26.2), 8 patients remain in remission, 4 have relapsed out of which 2 have died of progressive lymphoma. OS was 100% (95% CI: N/A) at 100-days, and89% (95% CI: 43-98) at 1 year. Non-relapse Mortality was 0% at both100-days and 1-year (95%CI: N/A) (95%CI: N/A). All patients successfully engrafted with the median days to ANC >= 500/ul was 11 (range: 10 - 12), and days to PLT >= 20,000/ul: 13 (12 - 92). No dose limiting toxicities were experienced. The most common/highest grade toxicity experienced (per Bearman Scale) was grade 2 stomatitis, which was seen in 3 patients at 0.4mCi/kg; 4 patients at 0.5 mCi/kg, and 3 at0.6mCi/kg. The only other toxicities seen were grade 2 GI in 2 patients at 0.4mCi/kg,and grade 2 bladder in one patient at 0.6mCi/kg dose.. Toxicities >grade 2 were not seen. Conclusion: aTac- BEAM appears to be safe as an ASCT conditioning regimen for PTCL with no increased toxicity as compared to the historical toxicities seen with BEAM alone in this patient population (D'Amore 2012 J of Clin Onc). The dose level 0.6mCi/kg will likely be the recommended phase II dose. An expanded phase is planned to evaluate the efficacy of this regimen followed by a randomized trial of BEAM alone plus a combination of aTac- BEAM. DisclosuresHerrera:Seattle Genetics: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Immune Design: Research Funding; KiTE Pharma: Consultancy, Research Funding; Merck, Inc.: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; AstraZeneca: Research Funding; Gilead Sciences: Research Funding. Salhotra:Kadmon Corporation, LLC: Consultancy.

The histologic, immunohistochemical, and genetic features of classical Hodgkin lymphoma and anaplastic large cell lymphoma with aberrant T-cell/B-cell antigen expression

Classical Hodgkin lymphoma (cHL) and ALK- anaplastic large cell lymphoma (ALCL) share many morphologic and immunohistochemical features, causing difficulties in differential diagnosis. Aberrant T-cell/B-cell antigen (TCA/BCA) expression in cHL/ALCL has previously been reported, but differences in the broader morphologic and genetic features still remain unclear. We first explored the histologic and immunohistochemical characteristics of cHL and ALCL with or without aberrant expression. Of 68 cHL cases, 10 (14.71%) were found to express 1 or more TCAs, and the frequency was as follows: CD4 > CD2 > CD3 > CD5 = CD7. Only 1 (3.33%) of 30 ALCL cases expressed BCA. Histologically, the main subtypes of cHL with aberrant TCA expression were LD and NS2. These aberrant TCA-expressing cHL tumor cells exhibited some ALCL features, and the aberrant BCA-expressing ALCL tumor cells displayed cHL characteristics. We also performed whole-exome sequencing analysis on cHL and ALCL samples with aberrant expression and compared them with those without aberrant expression. The results of this analysis showed that GNE and CACNB2 mutations, involved in the MAPK signaling pathway, may play an important role in cHL. In addition, 135 mutation sites involved in multiple signaling pathways were identified in ALCL. In the aberrant-expression cases, genetic features were similar between cHL and ALCL, consistent with their morphologic features. Our results broaden the understanding of the histologic and immunohistochemical characteristics of cHL and ALCL with aberrant expression and, for the first time, compare genetic features between cHL and ALCL with and without aberrant expression.

The Long Non-Coding RNA MIR503HG Enhances Proliferation of Human ALK-Negative Anaplastic Large-Cell Lymphoma.

Anaplastic lymphoma kinase (ALK)-negative anaplastic large-cell lymphoma (ALCL) is a rare type of highly malignant, non-Hodgkin lymphoma. Currently, only a few gene rearrangements have been linked to ALK-negative ALCL progression. However, the specific molecular mechanisms underlying the growth of ALK-negative ALCL tumors remain unclear. Here, we investigated aberrantly expressed, long non-coding RNAs (lncRNAs) in ALK-negative ALCL and assessed their potential biological function. MIR503HG (miR-503 host gene) was highly expressed in ALK-negative cell lines and was significantly upregulated in tumors in mice formed from ALK-negative ALCL cell lines. Depletion of MIR503HG suppressed tumor cell proliferation in vivo and in vitro; conversely, its overexpression enhanced tumor cell growth. MIR503HG-induced proliferation was mediated by the induction of microRNA-503 (miR-503) and suppression of Smurf2, resulting in stabilization of the tumor growth factor-β receptor (TGFBR) and enhanced tumor cell growth. Collectively, these findings support a potential role for MIR503HG in cancer cell proliferation through the miR-503/Smurf2/TGFBR axis and indicate that MIR503HG is a potential marker in ALK-negative ALCL.

Self-Assembled Aptamer-Nanomedicine for Targeted Chemotherapy and Gene Therapy.

Chemotherapy is the mainstream treatment of anaplastic large cell lymphoma (ALCL). However, chemotherapy can cause severe adverse effects in patients because it is not ALCL-specific. In this study, a multifunctional aptamer-nanomedicine (Apt-NMed) achieving targeted chemotherapy and gene therapy of ALCL is developed. Apt-NMed is formulated by self-assembly of synthetic oligonucleotides containing CD30-specific aptamer and anaplastic lymphoma kinase (ALK)-specific siRNA followed by self-loading of the chemotherapeutic drug doxorubicin (DOX). Apt-NMed exhibits a well-defined nanostructure (diameter 59 mm) and stability in human serum. Under aptamer guidance, Apt-NMed specifically binds and internalizes targeted ALCL cells. Intracellular delivery of Apt-NMed triggers rapid DOX release for targeted ALCL chemotherapy and intracellular delivery of the ALK-specific siRNA induced ALK oncogene silencing, resulting in combined therapeutic effects. Animal model studies reveal that upon systemic administration, Apt-NMed specifically targets and selectively accumulates in ALCL tumor site, but does not react with off-target tumors in the same xenograft mouse. Importantly, Apt-NMed not only induces significantly higher inhibition in ALCL tumor growth, but also causes fewer or no side effects in treated mice compared to free DOX. Moreover, Apt-NMed treatment markedly improves the survival rate of treated mice, opening a new avenue for precision treatment of ALCL.

NPM1‐ALK overexpression‐driven toxicity in ALCL is partner dependent and driven by phosphorylation of novel substrates leading to overstimulation of biosynthetic pathways

Introduction: Activation of the tyrosine-kinase domain of anaplastic lymphoma kinase (ALK) drives malignant growth in subsets of several cancers as part of oncogenic fusions resulting from chromosomal rearrangements. ALK stimulates several oncogenic pathways, including JAK/STAT3, MEK/ERK, and PI3K/AKT. Clinically, 70% of anaplastic large-cell lymphoma (ALCL) and ~5% of nonsmall-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 to 12 months is due to, most commonly, activation of alternate pathways or second-site kinase-domain mutations. We and others previously reported that, unlike NSCLC, ALCL tumor cells have great difficulty finding alternate pathways to replace ALK and instead upregulate expression of the NPM1-ALK fusion to maintain survival during inhibitor exposure. Strikingly, we found a toxic overdose of ALK signaling when inhibitors are withdrawn from overexpressing cells, triggering death. Here, we explore the mechanisms by which ALK overactivation drives cellular toxicity. Materials and Methods: We have generated multiple models of ALK+ ALCL with resistance to all generations ALK inhibitors. We employed RNAseq and phosphoproteomics to reveal candidate drivers of death upon ALK overexpression. We have generated multiple variant alleles of NPM1-ALK that enable us to interrogate the role of ALK's fusion partner in mediating these effects. Results: All generations of ALK inhibitors drive NPM1-ALK overexpression as the most frequent resistance mechanism in ALCL, but increased ALK activity upon inhibitor withdrawal is universally toxic. Extensive interrogations with targeted inhibitors and genetic approaches revealed that none of ALK's well-characterized downstream pathways is a key driver of overdose toxicity. Interestingly, while overexpression of NPM1-ALK confers selective disadvantage, elevated levels of activated ALK kinase domain alone do not. RNAseq analysis reveals hyperstimulation of protein biogenesis and RNA processing as cells die due to NPM1-ALK overdose. Phosphoproteomics results align with RNAseq data showing increased phosphorylation of proteins driving the same pathways. Taken together our results show the NPM1 translocation partner plays a key role driving NPM1-ALK overdose in ALCL by promoting phosphorylation of novel substrates. Conclusions: In contrast to ALK+ NSCLC, ALCL cannot easily bypass a need for ALK by activating alternate pathways. NPM1-ALK overexpression-driven toxicity is partner dependent, driving death associated with hyperactivation of protein and RNA processing pathways. Drugs that drive ALK toward phosphorylation of noncanonical substrates therefore represent a novel therapeutic strategy worth pursuing in ALCL and other ALK-driven malignancies. Keywords: ALK; anaplastic large cell lymphoma (ALCL)

A Pharmacologically Reversible Epigenomic Signature of Relapse and Chemotherapy Resistance in Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) are orphan entities representing around 20% of all Peripheral T-cell lymphoma. ALCLs can be stratified into primary cutaneous ALCL and systemic ALCL. Patients with systemic ALCL are further distinguished based on anaplastic lymphoma kinase (ALK) translocations. Unlike ALK+ patients, ALK- ALCLs are more likely to be refractory or relapse upon treatment with CHOP (Cyclophosphamide, Hydroxydaunorubicin, Oncovin, Prednisone). A small number of genetic defects and genomic translocations have been identified and associated with clinical outcome in ALK- ALCL, but cannot fully explain the poor outcomes of ALK- patients. To further probe the molecular bases of ALCL, we performed systematic epigenomic profiling in systemic ALCL through Enhanced Reduced Representation Bisulfite Sequencing (ERRBS).To investigate the methylation profile of systemic ALCLs, we first interrogated 8 ALK+ ALCL and 16 ALK- ALCL tumors. Combining methylation levels from all interrogated CpG sites, we observed overall hypermethylation at CpG-islands (CGIs; p=0.02, t-test) in ALK- tumors compared ALK+ patients but hypomethylation outside of CGIs (>10kb away from known CGIs; p=0.03, t-test). There were no significant differences on the average methylation levels at CpG-island shores, which indicate varied methylation profiles among different genomic locations. To define methylation-based signatures, we then performed supervised analysis between ALK+ and ALK- patients. We have identified 254 ALK- patients linked consistently differentially methylated promoters (>20% DNA methylation changes and FDR<0.1, t-test). Out of 254 promoters, 252 genes were hypermethylated in ALK- patients confirming the association between systemic ALCL subtypes and hypermethylation.We then sought to investigate how methylation evolves in ALCL. We performed ERRBS of 2 primary ALCL tumors and their matched relapse samples (2 for each patient). We observed a significant hypermethylation at CGIs in relapsed samples (p=1.4e-5, paired t-test), suggesting that hypermethylation changes may be associated with tumor evolution.To test whether the hypermethylation of specific genes is linked to tumor aggressiveness, we analyzed serial passages of patient-derived tumor xenograft (PDTX) derived from primary and relapse ALCL samples. We observed consistently hypermethylation at CGIs along different implants and conserved hypermethylation of genes at the same time (p<0.05, 1959). To confirm the behavior of those genes, we estimate their methylation trend in ALCL primary and relapse samples with GSEA. Target genes showed a very strong hypermethylation in relapsed ALCL compared to primary samples (FDR<0.01). Next we confirmed analogous methylation changes in a cohort of primary and relapse PDTX (Primary, n=2, FDR<0.01; Relapse, n=3, FDR<0.01). Interestingly, when comparing this to our previous diffuse large b-cell lymphoma (DLBCL) study (Pan et.al., 2015), we found similar hypermethylated profiles, suggesting commonalities among different type of human lymphomas.Lastly, we investigated whether a treatment with the 5-azacytidine (5-Aza) could reverse the hypermethylation profile of ALCL PDTX. Tumors from in vivo 5-Aza treated and control mice were extracted and subjected to ERRBS. As expected, we only found global hypomethylation in 5-Aza-treated samples closely mimicked methylation profiles in naive primary ALCL and early passages of PDTX.In summary, this study provides the first comprehensive characterization of methylome in ALCL. Hypermethylation is firstly associated with the aggressiveness of ALK- patients in ALCL. Also, we identified a group of hypermethylated genes associated with relapsed ALCL, a phenotype mirrored by serial passages of PDTX in vivo. As 5-Aza treatments can revert the methylome of late passages of ALCL PDTX, we speculate that this treatment may represent a novel therapeutic approach in chemo-relapsed ALCL patients. DisclosuresMartin:Gilead: Consultancy, Other: travel, accommodations, expenses; Acerta: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Honoraria; Teva: Research Funding; Janssen: Consultancy, Honoraria, Other: travel, accommodations, expenses.

Insights into the Pathogenesis of Anaplastic Large-Cell Lymphoma through Genome-wide DNA Methylation Profiling

SummaryAberrant DNA methylation patterns in malignant cells allow insight into tumor evolution and development and can be used for disease classification. Here, we describe the genome-wide DNA methylation signatures of NPM-ALK-positive (ALK+) and NPM-ALK-negative (ALK−) anaplastic large-cell lymphoma (ALCL). We find that ALK+ and ALK− ALCL share common DNA methylation changes for genes involved in T cell differentiation and immune response, including TCR and CTLA-4, without an ALK-specific impact on tumor DNA methylation in gene promoters. Furthermore, we uncover a close relationship between global ALCL DNA methylation patterns and those in distinct thymic developmental stages and observe tumor-specific DNA hypomethylation in regulatory regions that are enriched for conserved transcription factor binding motifs such as AP1. Our results indicate similarity between ALCL tumor cells and thymic T cell subsets and a direct relationship between ALCL oncogenic signaling and DNA methylation through transcription factor induction and occupancy.

The NADPH oxidase NOX5 protects against apoptosis in ALK-positive anaplastic large-cell lymphoma cell lines

Reactive oxygen species (ROS) are key modulators of apoptosis and carcinogenesis. One of the important sources of ROS is NADPH oxidases (NOXs). The isoform NOX5 is highly expressed in lymphoid tissues, but it has not been detected in any common Hodgkin or non-Hodgkin lymphoma cell lines. In diverse, nonlymphoid malignant cells NOX5 exerts an antiapoptotic effect. Apoptosis suppression is the hallmark feature of a rare type of lymphoma, termed anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL), and a major factor in the therapy resistance and relapse of ALK+ ALCL tumors. We applied RT-PCR and Western blot analysis to detect NOX5 expression in three ALK+ ALCL cell lines (Karpas-299, SR-786, SUP-M2). We investigated the role of NOX5 in apoptosis by small-interfering RNA (siRNA)-mediated gene silencing and chemical inhibition of NOX5 using FACS analysis and examining caspase 3 cleavage in Karpas-299 cells. We used immunohistochemistry to detect NOX5 in ALK+ ALCL pediatric tumors. NOX5 mRNA was uniquely detected in ALK+ ALCL cells, whereas cell lines of other lymphoma classes were devoid of NOX5. Transfection of NOX5-specific siRNA and chemical inhibition of NOX5 abrogated calcium-induced superoxide production and increased caspase 3-mediated apoptosis in Karpas-299 cells. Immunohistochemistry revealed focal NOX5 reactivity in pediatric ALK+ ALCL tumor cells. These results indicate that NOX5-derived ROS contribute to apoptosis blockage in ALK+ ALCL cell lines and suggest NOX5 as a potential pharmaceutical target to enhance apoptosis and thus to suppress tumor progression and prevent relapse in pediatric ALK+ ALCL patients that resist classical therapeutic approaches.

Primary Gastric Anaplastic Lymphoma Kinase (ALK) Negative, Large-Cell Lymphoma Causing Gastrointestinal Bleeding in a Patient With Roux-en-Y Gastric Bypass

Introduction: Anaplastic large cell lymphoma (ALCL) is a rare, CD30 positive T-cell neoplasm accounting for 3% of all non-Hodgkin’s lymphomas. Primary lesions are rarely found in the gastrointestinal (GI) tract. Patients may present with dyspepsia, bleeding, etc. Treatment usually requires resection with adjuvant chemotherapy. Even with treatment, the anaplastic lymphoma kinase negative (ALK-negative) variant has a 30-49% 5-year survival rate. This is the case of a 54-year-old female with a history of Rouxen-Y gastric bypass presenting with severe GI bleeding due to reoccurrence of a primary ALK-negative ALCL tumor in the gastric remnant. This lead to hemorrhagic ischemic necrosis, perforation, and splenic invasion, requiring surgical intervention. Remnant perforation is rare, reported mainly in case reports. Endoscopy should be performed to rule out anastomotic ulcerations, and deep enteroscopy if remnant bleeding is suspected. Increased understanding of a bariatric patient’s altered anatomy and adequate cancer surveillance may improve outcomes if complications are recognized early on in these patients.Figure 1: Anastomotic ulcer actively bleeding.Figure 2: Anastomotic ulcer after hemostasis.Figure 3: Immunohistochemical stain for CD30 showing infiltrative cells in the splenic tumor section.

A cancer cell-activatable aptamer-reporter system for one-step assay of circulating tumor cells.

The current antibody-mediated numeration assays of circulating tumor cells (CTCs) require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

RIP1 expression is necessary for CD30-mediated cell death induction in anaplastic large-cell lymphoma cells

CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, is consistently expressed by tumor cells of anaplastic large-cell lymphoma (ALCL). CD30 stimulation induces massive caspase-dependent cell death of ALCL cells in case of canonical NFκB inhibition or proteasome inhibition. However, CD30, a TNFR lacking a death domain (DD), is unable to recruit a death inducing complex containing TRADD (TNFR1-associated DD-protein) or FADD (FAS-associated DD-domain protein) together with the receptor-interacting protein 1 (RIP1) and caspase-8. Thus, the mechanism explaining CD30-induced cell death of lymphocytes remains obscure. Here, we demonstrate that blockage of RIP1 by siRNA or pharmacological inhibition of RIP1 by Necrostatin-1 almost completely prevented CD30-induced cell death. In addition, we revealed CD30-induced accumulation of RIP1 at the cytoplasma membrane of NFκB-inhibited ALCL cells by confocal laser scanning microscopy. Finally, primary ALCL cases can be subdivided into two groups based on the presence or absence of RIP1 as revealed by immunohistology. Taken together, our study identified RIP1 as a crucial mediator of CD30-induced cell death that bears features of apoptosis as well as necroptosis. RIP1 expression in ALCL tumor cells might eligible for the therapeutic application of CD30 antibodies in combination with NFκB/proteasome inhibitors that should result in CD30-induced cell death.

Combinatorial Effects of CD30 Ligand Stimulation or Inhibitor of Apoptosis (IAP) Antagonist Exposure with Conventional Chemotherapy on CD30 Positive Malignancies

AbstractAbstract 4926CD30 is a member of the tumor necrosis factor (TNF) receptor family that is normally found on the cell surface of a small subset of activated lymphocytes but is overexpressed on the surface of anaplastic large cell lymphoma (ALCL) and Hodgkins lymphoma (HL) cells. Although many drugs exist that treat lymphomas by triggering the intrinsic cell death pathway, current chemotherapeutic regimens are limited by unwanted side effects, including secondary malignancies that limit event-free survival. The tumor-restricted overexpression of CD30 makes it an attractive target for therapeutic intervention. Depending on the cellular context, CD30 stimulation has been linked to cell death, cell cycle arrest, or paradoxically, proliferation. In ALCL tumor cell lines, CD30 stimulation activates both the canonical and noncanonical NF-kB pathways while in HL tumor cell lines, CD30 stimulation only slightly enhances NF-kB activity above constitutive levels, implying a role for NF-kB in determining the sensitivity or resistance of lymphoma cells to CD30-induced apoptosis. In addition, IAP antagonists, small synthetic compounds that mimic the structure of the second mitochondrial activator of caspase (Smac) and target IAP molecules that affect the activation of the non-canonical NF-kB pathway, induce apoptosis and/or sensitize cells to death via secondary signals such as TNF. This suggests that the modulation of IAP levels, and consequently regulation of the non-canonical NF-kB pathways, may also have a role in determining tumor cell death. Using representative ALCL and HL tumor cell lines, we have found that CD30 stimulation via its physiologically ligand in combination with standard chemotherapeutic agents results in increased efficacy in tumor cell death in the majority of ALCL cell lines but not HL cell lines. Similarly, IAP antagonists in combination with standard chemotherapeutic agents also resulted in enhanced tumor cell death in most ALCL but not HL cell lines. This augmentation of tumor cell death suggests that CD30-induced apoptosis and IAP antagonist-induced killing may have important consequences in the clinical treatment of CD30 positive malignancies. Currently, we are further investigating the role of both CD30 stimulation via its physiological ligand and IAP antagonists in impacting the activation of the canonical and noncanonical NF-kB pathways alone and in combination with currently utilized chemotherapeutic agents to modulate the apoptotic threshold in CD30 positive lymphoma cells. Disclosures:No relevant conflicts of interest to declare.

Discovery of a potent inhibitor of anaplastic lymphoma kinase with in vivo antitumor activity.

A series of novel 7-amino-1,3,4,5-tetrahydrobenzo[b]azepin-2-one derivatives within the diaminopyrimidine class of kinase inhibitors were identified that target anaplastic lymphoma kinase (ALK). These inhibitors are potent against ALK in an isolated enzyme assay and inhibit autophosphorylation of the oncogenic fusion protein NPM-ALK in anaplastic large cell lymphoma (ALCL) cell lines. The lead inhibitor 15, which incorporates a bicyclo[2.2.1]hept-5-ene ring system in place of an aryl moiety, activates the pro-apoptotic caspases (3 and 7) and displays selective cytotoxicity against ALK-positive ALCL cells. Furthermore, 15 provides more than 40-fold selectivity against the structurally related insulin receptor, is orally bioavailable in multiple species, and displays in vivo antitumor efficacy when dosed orally in ALK-positive ALCL tumor xenografts in Scid mice.