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Supplementary Figure S1. ALK Tg mice develop lung multifocal adenocarcinomas similar to human ALK-rearranged NSCLC. Supplementary Figure S2. Therapeutic ALK vaccine impairs tumor growth of ALK-rearranged lung tumors. Supplementary Figure S3. Immunophenotype of EML4-ALK transgenic mice Supplementary Figure S4. Human ALK-rearranged NSCLC show a pattern of low expression of T cell markers. Supplementary Figure S5. PD-L1 expression is dependent on ALK kinase activity in ALK-rearranged NSCLC cells. Supplementary Figure S6. Schematic protocols of the in vivo treatment with anti-PD-1 blocking antibody. Supplementary Figure S7. ALK vaccine can be combined with ALK inhibitor TAE684. Supplementary Figure S8. Immunophenotype of intratumoral T cells in ALK vaccinated mice.

Supplementary Figure Legends

Supplementary Materials and Methods

Non–small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKI), but the treatment is successful for only a limited amount of time; most patients experience a relapse due to the development of drug resistance. Here, we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti–PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC. Cancer Immunol Res; 3(12); 1333–43. ©2015 AACR.

Supplementary Tables S1-S7. K-RasG12V tumors serially transplanted in Crl:NU-Foxn1nu mice and paired derived cell lines (S1); Semiquantitative assessment of the percentages of histologic components in passage #1 engrafted tumors (S2); EML4-ALK tumors orthotopically implanted in Crl:NU-Foxn1nu mice (S3); Histologic progression of orthoallografts upon sequential mice-to-mice passages (S4); Presence of distal metastasis in animals implanted with the indicated orthoallografts (S5); Summary of primary cell lines obtained from the indicated orthoallograft implants (S6); Histopathological characterization of EML4-ALK implants after crizotinib treatment (S7).

Supplementary Figures 1-19. Supplementary Figure 1. Hypoxia pathways are enriched in ALCL. Supplementary Figure 2. Gene expression data on ALCL. Supplementary Figure 3. EML4-ALK NSCLC show enrichment in hypoxia pathway associated genes. Supplementary Figure 4. ALK regulates HIF-1α and HIF-2α expression in ALK-rearranged ALCL cell lines. Supplementary Figure 5. Oncogenic ALK regulates HIF-1α and HIF-2α expression levels in ALK-rearranged ALCL cell lines. Supplementary Figure 6. Oncogenic ALK-mediated control of HIF-1α and HIF-2α expression is not dependent on proteasome-dependent degradation. Supplementary Figure 7. ALK regulates HIF-1α and HIF-2α through STAT3 and C/EBPβ. Supplementary Figure 8. STAT3 and C/EBPβ mediated regulation of HIFs in ALCL. Supplementary Figure 9. Ectopic expression of NPM-ALK in ALK negative ALCL. Supplementary Figure 10. Validation of shRNA against HIF-1α and HIF-2α. Supplementary Figure 11. HIF-2α is essential for the growth of ALK-rearranged ALCL in vivo. Supplementary Figure 12. HIF-1α and HIF-2α are not required for the growth of ALK-negative T cell lymphoma in vivo. Supplementary Figure 13. ALK regulates VEGFA mRNA expression and production in vitro. Supplementary Figure 14. Anti-angiogenic treatment in ALK negative ALCL. Supplementary Figure 15. Oncogenic ALK regulates HIF-1α and HIF-2α expression levels in EML4-ALK NSCLC. Supplementary Figure 16. HIF-α regulation in NSCLC is specifically dependent on ALK activity. Supplementary Figure 17. Oncogenic ALK activity up-regulates VEGF expression in NSCLC. Supplementary Figure 18. HIF-α knock-down does not affect the growth of K-RAS mutated NSCLC. Supplementary Figure 19. HIF-1α and HIF-2α are essential for metastasis formation in EML4-ALK NSCLC.

Supplementary Figure Legends. Legend for Supplementary Figures S1-S4.

Supplementary Figures S1-S4. Common histological subtypes of NSCLC orthoallografts (S1); In vitro characterization of primary cell lines derived from NSCLC orthoallografts (S2); Lung colonization and tumor formation upon reintroduction of mKLC cell lines (S3); Inoculation of mKLC single cell clones gives rise to NSCLC heterogeneity in vivo (S4).

Supplementary Figure Legends. File with Supplementary Figure Legends

FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML.

Ruxolitinib (Jakavi) is a potent JAK1/JAK2 specific inhibitor, approved for the treatment of myeloproliferative neoplasia (MPN) such as primary myelofibrosis (PMF) and polycythemia Vera (PV). The primary clinical benefit of ruxolitinib in MPN patients is reduction in spleen size and alleviation of constitutional symptoms and significant improvement in the quality of life. However, the effect of ruxolitinib on bone marrow fibrosis and JAK2V617F allelic burden is modest. In addition, JAK2V617F negative MPNs also demonstrated clinical benefits, thus, is not clear whether the ruxolitinib primarily blocks the proliferation of the malignant clone or exerts its effects by targeting non-malignant cells. To gain the mechanism of ruxolitinib action in MPNs, we developed two JAK2V617F-driven MPN mouse models harboring ruxolitinib resistant mutations JAK2V617F+L902Q and JAK2V617F+L983F in the malignant clone. Similar to JAK2V617F recipients, JAK2V617F+L902Q and JAK2V617F+L983F transplanted mice showed an increase in WBC, HCT, RBC, HB and reticulocyte values. Histopathological analysis revealed that the myeloproliferative phenotype resembles PV with a proliferation of all three lineages albeit with only a moderate increase in megakaryopoeisis. Grade II bone marrow fibrosis was observed in JAK2V617F+L902Q and JAK2V617F+L983F after 2-3 months similar to JAK2V617F mice. Surprisingly, these mice respond to ruxolitinib treatment similar to ruxolitinib sensitive JAK2V617F mice, indicated by reduction of spleen size, leukocyte count and proinflammatory cytokines in the serum. Mechanistically, we could identify a direct role of ruxolitinib in the impairment of inflammatory processes in the bone marrow microenvironment, particularly on the cytokine production of mesenchymal stem cells and endothelial cells. These results suggest that the ruxolitinib mediated effects in MPN patients are mainly due to abrogation of inflammatory signaling processes in non-malignant cells. Therefore, combination treatment of JAK1/2 inhibitors with novel inhibitors, which specifically act on malignant cells might improve the clinical benefit for MPN patients to JAK family inhibitors. Citation Format: Sivahari Prasad Gorantla, Michael Rassner Rassner, Tony Andreas Müller, Teresa Poggio, Kirstyn Anne Crossley, Anna-Lena Denecke, Kornelia R Fritsch, Geoffroy Andrieux, Helen Kleinfelder, Shifa Khaja Saleem, Sudheer Madan Mohan Gambheer, Irene Gonzalez Menendez, Detlef Bentrop, Rainer Trittler, Svetlana Rylova, Dietmar Pfeifer, Leticia Quintanilla Martinez, Christine Dierks, Robert Zeiser, Anna Lena Illert, Nikolas von Bubnoff, Justus Duyster. Efficacy of Jak1/2 inhibition in murine myeloproliferative neoplasms is mediated by targeting nonmalignant cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4497.

Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.

Internal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) predict poor prognosis in acute myeloid leukemia (AML) and often co-exist with inactivating DNMT3A mutations. In vitro studies implicated Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. Utilizing a Flt3-ITD knock-in, Dnmt3a haploinsufficient mouse model, we demonstrate that Gab2 is essential for the development of Flt3-ITD driven AML in vivo, as Gab2 deficient mice displayed prolonged survival, presented with attenuated liver and spleen pathology and reduced blast counts. Furthermore, leukemic bone marrow from Gab2 deficient mice exhibited reduced colony-forming unit capacity and increased FLT3 inhibitor sensitivity. Using transcriptomics, we identify the genes encoding for Axl and the Ret co-receptor Gfra2 as targets of the Flt3-ITD/Gab2/Stat5 axis. We propose a pathomechanism in which Gab2 increases signaling of these receptors by inducing their expression and by serving as downstream effector. Thereby, Gab2 promotes AML aggressiveness and drug resistance as it incorporates these receptor tyrosine kinases into the Flt3-ITD signaling network. Consequently, our data identify GAB2 as a promising biomarker and therapeutic target in human AML.

FLT3-ITD is the most predominant mutation in AML being expressed in about one-third of AML patients and is associated with a poor prognosis. Efforts to better understand FLT3-ITD downstream signaling to possibly improve therapy response are needed. We have previously described FLT3-ITD-dependent phosphorylation of CSF2RB, the common receptor beta chain of IL-3, IL-5, and GM-CSF, and therefore examined its significance for FLT3-ITD-dependent oncogenic signaling and transformation. We discovered that FLT3-ITD directly binds to CSF2RB in AML cell lines and blasts isolated from AML patients. A knockdown of CSF2RB in FLT3-ITD positive AML cell lines as well as in a xenograft model decreased STAT5 phosphorylation, attenuated cell proliferation, and sensitized to FLT3 inhibition. Bone marrow from CSF2RB-deficient mice transfected with FLT3-ITD displayed decreased colony formation capacity and delayed disease onset together with increased survival upon transplantation into lethally irradiated mice. FLT3-ITD-dependent CSF2RB phosphorylation required phosphorylation of the FLT3 juxtamembrane domain at tyrosines 589 or 591, whereas the ITD insertion site and sequence were of no relevance. Our results demonstrate that CSF2RB participates in FLT3-ITD-dependent oncogenic signaling and transformation in vitro and in vivo. Thus, CSF2RB constitutes a rational treatment target in FLT3-ITD-positive AML.

Targeted expression of transgenes is essential for the accurate representation of human disease in in vivo models. Current approaches to generate conditional transgenic mouse models are cumbersome and not amenable to high-throughput analysis since they require de novo generation and characterization of genetically modified mice. Here we describe a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is achieved by retroviral infection and transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage-specific promoter. For validation, we directed expression of NPM-ALK, the fusion oncogene driving a subset of anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre-transgenic mice with a retroviral construct containing the NPM-ALK cDNA preceded by a translational stop cassette. These mice developed T-cell lymphomas within 12-16 weeks, featuring increased expression of the ALCL hallmark antigen CD30 as well as other cytotoxic T-cell markers, similar to the human disease. The new model represents a versatile tool for the rapid analysis of gene function in a defined lineage or in a developmental stage in vivo.

Internal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) frequently co-exist with inactivating DNMT3A mutations and predict poor prognosis in acute myeloid leukemia (AML). In vitro studies implicated the docking protein Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. By utilizing a Flt3-ITD knock-in, Dnmt3a haploinsufficient mouse model, we demonstrate that Gab2 is essential for the development of Flt3-ITD driven AML in vivo, as Gab2 deficient mice survived significantly longer, had reduced blast counts and exhibited an attenuated spleen and liver pathology as compared to their Gab2-proficient littermates. Further, leukemic bone marrow isolated from Gab2 deficient mice displayed a reduced colony forming unit capacity and an increased sensitivity towards the Flt3 inhibitor quizartinib. Using RNA sequencing, we identify the genes encoding for Axl and the Ret co-receptor Gfra2 as targets of the Flt3-ITD/Gab2/Stat5 axis. We propose a pathomechanism in which Gab2 increases signaling of these receptors as it induces their expression and additionally serves as a downstream effector. Consequently, Gab2 promotes AML aggressiveness as well as drug resistance by incorporating the receptor tyrosine kinases Axl and Ret into the Flt3-ITD signaling network. Therefore, our data identify GAB2 as a promising biomarker as well as a therapeutic target in human AML. Citation Format: Corinna Spohr, Teresa Poggio, Geoffroy Andrieux, Katharina Schönberger, Nina Cabezas-Wallscheid, Melanie Börries, Sebastian Halbach, Anna L. Illert, Tilman Brummer. Gab2 deficiency protects against Flt3-ITD driven acute myeloid leukemia in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 945.

The expression of BCL6 in B-cell lymphoma can be deregulated by chromosomal translocations, somatic mutations in the promoter regulatory regions, or reduced proteasome-mediated degradation. FBXO11 was recently identified as a ubiquitin ligase that is involved in the degradation of BCL6, and it is frequently inactivated in lymphoma or other tumors. Here, we show that FBXO11 mutations are found in 23% of patients with Burkitt lymphoma (BL). FBXO11 mutations impaired BCL6 degradation, and the deletion of FBXO11 protein completely stabilized BCL6 levels in human BL cell lines. Conditional deletion of 1 or 2 copies of the FBXO11 gene in mice cooperated with oncogenic MYC and accelerated B-cell lymphoma onset, providing experimental evidence that FBXO11 is a haploinsufficient oncosuppressor in B-cell lymphoma. In wild-type and FBXO11-deficient BL mouse and human cell lines, targeting BCL6 via specific degraders or inhibitors partially impaired lymphoma growth in vitro and in vivo. Inhibition of MYC by the Omomyc mini-protein blocked cell proliferation and increased apoptosis, effects further increased by combined BCL6 targeting. Thus, by validating the functional role of FBXO11 mutations in BL, we further highlight the key role of BCL6 in BL biology and provide evidence that innovative therapeutic approaches, such as BCL6 degraders and direct MYC inhibition, could be exploited as a targeted therapy for BL.

Anaplastic large-cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma that shows in 60% of cases a translocation t(2, 5)(p23, q35), which leads to the expression of the oncogenic kinase NPM-ALK. The nuclear interaction partner of ALK (NIPA) defines an E3-SCF ligase that contributes to the timing of mitotic entry. It has been shown that co-expression of NIPA and NPM-ALK results in constitutive NIPA phosphorylation. By mass spectrometry-based proteomics we identified nine serine/threonine residues to be significantly upregulated in NIPA upon NPM-ALK expression. Generation of phospho-deficient mutants of the respective phospho-residues specified five serine/threonine residues (Ser-338, Ser-344, Ser-370, Ser-381 and Thr-387) as key phosphorylation sites involved in NPM-ALK-directed phosphorylation of NIPA. Analysis of the biological impact of NIPA phosphorylation by NPM-ALK demonstrated that the ALK-induced phosphorylation does not change the SCFNIPA-complex formation but may influence the localization of NIPA and NPM-ALK. Biochemical analyses with phospho-deficient mutants elucidated the importance of NIPA phosphorylation by NPM-ALK for the interaction of the two proteins and proliferation potential of respective cells: Silencing of the five crucial NIPA serine/threonine residues led to a highly enhanced NIPA-NPM-ALK binding capacity as well as a slightly reduced proliferation in Ba/F3 cells.

Activating mutations in FMS-like tyrosine kinase receptor-3 (FLT3) and Nucleophosmin-1 (NPM1) are most frequent alterations in acute myeloid leukemia (AML), and are often coincidental. The mutational status of NPM1 has strong prognostic relevance to patients with point mutations of the FLT3 tyrosine kinase domain (TKD), but the biological mechanism underlying this effect remains unclear. In the present study, we investigated the effect of the coincidence of NPM1c and FLT3-TKD. Although expression of FLT3-TKD is not sufficient to induce a disease in mice, coexpression with NPM1c rapidly leads to an aggressive myeloproliferative disease in mice with a latency of 31.5 days. Mechanistically, we could show that FLT3-TKD is able to activate the downstream effector molecule signal transducer and activator of transcription 5 (STAT5) exclusively in the presence of mutated NPM1c. Moreover, NPM1c alters the cellular localization of FLT3-TKD from the cell surface to the endoplasmic reticulum, which might thereby lead to the aberrant STAT5 activation. Importantly, aberrant STAT5 activation occurs not only in primary murine cells but also in patients with AML with combined FLT3-TKD and NPM1c mutations. Thus, our data indicate a new mechanism, how NPM1c mislocalizes FLT3-TKD and changes its signal transduction ability.

While cancer stem cells are well established in certain hematologic and solid malignancies, their existence in T cell lymphoma is unclear and the origin of disease is not fully understood. To examine the existence of lymphoma stem cells, we utilized a mouse model of anaplastic large cell lymphoma. Established NPM-ALK+ lymphomas contained heterogeneous cell populations ranging from mature T cells to undifferentiated hematopoietic stem cells. Interestingly, CD4−/CD8− double negative (DN) lymphoma cells aberrantly expressed the T cell receptor α/β chain. Serial transplantation of sorted CD4/CD8 and DN lymphoma subpopulations identified lymphoma stem cells within the DN3/DN4 T cell population, whereas all other subpopulations failed to establish serial lymphomas. Moreover, transplanted lymphoma DN3/DN4 T cells were able to differentiate and gave rise to mature lymphoma T cells. Gene expression analyses unmasked stem-cell-like transcriptional regulation of the identified lymphoma stem cell population. Furthermore, these lymphoma stem cells are characterized by low CD30 expression levels, which might contribute to limited long-term therapeutic success in patients treated with anti-CD30-targeted therapies. In summary, our results highlight the existence of a lymphoma stem cell population in a NPM-ALK-driven CD30+ mouse model, thereby giving the opportunity to test innovative treatment strategies developed to eradicate the origin of disease.

In T lymphocytes, the Wiskott–Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase–positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-β. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL., The work has been supported by grant no. FP7 ERC-2009-StG (Proposal No. 242965—‘Lunely’) (R.C.) grant no. R01 CA196703-01 (R.C.); AIRC grant no. MFAG (C.A. and M.C.); National Research Foundation of Korea (NRF) fellowship 2016R1A6A3A03006840 (T-C.C.); Bando Giovani Ricercatori grant no. 2009-GR 1603126 (M.C.); MINECO/FEDER grant no. SAF2015–70368-R and Fundación Ramón Areces (I.M.A.); the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health (L.D.N.); and award no. T32GM007753 from the National Institute of General Medical Sciences (S.H.C.) (the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health); and in part by awards from the National Institutes of Health DP2 New Innovator award no. 1DP2CA195762-01 (C.K.); the American Cancer Society Research Scholar award no. RSG-14-051-01-DMC and the Pew-Stewart Scholars in Cancer Research Grant (C.K.); and the European Union Horizon 2020 Marie Sklodowska-Curie Innovative Training Network Grant award no. 675712 for the European Research Initiative for ALK-Related Malignancies (G.G.S., I.M., C.GP. and R.C.).

Targeted expression of transgenes is essential for the accurate representation of human disease in in vivo models. Current approaches to generate conditional transgenic mouse models are cumbersome and not amenable to high-throughput analysis since they require de novo generation and characterization of genetically modified mice. Here we describe a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is achieved by retroviral infection and transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage-specific promoter. For validation, we directed expression of NPM-ALK, the fusion oncogene driving a subset of anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre-transgenic mice with a retroviral construct containing the NPM-ALK cDNA preceded by a translational stop cassette. These mice developed T-cell lymphomas within 12-16 weeks, featuring increased expression of the ALCL hallmark antigen CD30 as well as other cytotoxic T-cell markers, similar to the human disease. The new model represents a versatile tool for the rapid analysis of gene function in a defined lineage or in a developmental stage in vivo.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F–myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient–derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1–mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive PD-L1 expression may help in developing new therapeutic strategies. Here, we show that oncogenic JAK2 activity caused STAT3 and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2(V617F)-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2(V617F)-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2(V617F)-myeloproliferative neoplasms (MPN) compared to healthy individuals and declined upon JAK2 inhibition. JAK2(V617F) mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2(V617F)-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2(V617F)-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Rearrangements involving the anaplastic lymphoma kinase (ALK) gene are defining events in several tumors, including anaplastic large-cell lymphoma (ALCL) and non–small cell lung carcinoma (NSCLC). In such cancers, the oncogenic activity of ALK stimulates signaling pathways that induce cell transformation and promote tumor growth. In search for common pathways activated by oncogenic ALK across different tumors types, we found that hypoxia pathways were significantly enriched in ALK-rearranged ALCL and NSCLC, as compared with other types of T-cell lymphoma or EGFR- and K-RAS–mutated NSCLC, respectively. Consistently, in both ALCL and NSCLC, we found that under hypoxic conditions, ALK directly regulated the abundance of hypoxia-inducible factors (HIF), which are key players of the hypoxia response in normal tissues and cancers. In ALCL, the upregulation of HIF1α and HIF2α in hypoxic conditions required ALK activity and its downstream signaling proteins STAT3 and C/EBPβ. In vivo, ALK regulated VEGFA production and tumor angiogenesis in ALCL and NSCLC, and the treatment with the anti-VEGFA antibody bevacizumab strongly impaired ALCL growth in mouse xenografts. Finally, HIF2α, but not HIF1α, was required for ALCL growth in vivo whereas the growth and metastasis potential of ALK-rearranged NSCLC required both HIF1α and HIF2α. In conclusion, we uncovered an ALK-specific regulation of the hypoxia response across different ALK+ tumor types and propose HIFs as a powerful specific therapeutic target in ALK-rearranged ALCL and NSCLC. Cancer Res; 74(21); 6094–106. ©2014 AACR.

Cancer evolution is a process that is still poorly understood because of the lack of versatile in vivo longitudinal studies. By generating murine non–small cell lung cancer (NSCLC) orthoallobanks and paired primary cell lines, we provide a detailed description of an in vivo, time-dependent cancer malignization process. We identify the acquisition of metastatic dissemination potential, the selection of co-driver mutations, and the appearance of naturally occurring intratumor heterogeneity, thus recapitulating the stochastic nature of human cancer development. This approach combines the robustness of genetically engineered cancer models with the flexibility of allograft methodology. We have applied this tool for the preclinical evaluation of therapeutic approaches. This system can be implemented to improve the design of future treatments for patients with NSCLC. Cancer Res; 74(21); 5978–88. ©2014 AACR.

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by impaired stem cell function resulting in pancytopenia. Diagnosis of IBMFS presents a major challenge due to limited diagnostic tests and overlapping phenotypes. For that reason, novel and clinical relevant biomarkers and possible targets are urgently needed. Our study defines NIPA as an IBMFS gene, which is significantly downregulated in a distinct subset of MDS-type refractory cytopenia of childhood patients. Mechanistically, NIPA binds FANCD2 and regulates its nuclear abundance. The stabilization of both non- and monoubiquitinated FANCD2 identifies NIPA as an essential player in the Fanconi Anemia (FA) pathway. NIPA thereby prevents MMC hypersensitivity visualized by increased numbers of chromosome radials and reduced cell survival after induction of interstrand crosslinks. To provide proof of principle, re-expression of FANCD2 in Nipa deficient cells restores MMC sensitivity. In a knockout mouse model, Nipa deficiency leads to major cell intrinsic long-term repopulation defects of hematopoietic stem cells (HSCs), with impaired self-renewal in serial transplantations and a bias towards myeloid differentiation. Unresolved DNA damage in Nipa deficient HSCs causes increased sensitivity to cell death and leads to progressive, age-related loss of the HSC pool. Induction of replication stress triggers the phenotypic reduction and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the mice thereby mimicing Fanconi Anemia. Taken together, our study adds NIPA to the short list of FA-associated proteins being essential for a functional DNA repair/FA/BRCA axis and thereby emphasizing its impact as potential diagnostic marker and/or possible target in bone marrow failure syndromes. Disclosures Niemeyer: Celgene: Consultancy.

Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of T-cell non-Hodgkin lymphoma (NHL) mainly affecting children and young adults. About 70% of systemic ALCLs are associated with a characteristic chromosomal translocation, t(2;5)(p23;35) which fuses the anaplastic lymphoma kinase (ALK) gene on chromosome 2 with the nucleophosmin (NPM) gene on chromosome 5, resulting in the NPM-ALK fusion gene, its over-expression and constitutive kinase activity. Immunophenotypic characterization of human ALCL revealed highly CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical trials with CD30-coupled antibodies. However, the impact of CD30 on disease development remains unclear and the relationship between NPM-ALK and CD30 has been investigated mostly using cell line models. Syngeneic mouse models of cancer can serve as useful models since the tumors develop in situ where the contribution made by the immune system and the extracellular matrix can be investigated. Here, we focus on the involvement of CD30 in a retroviral murine bone marrow transplantation model of ALCL. In this model, the BM of Lck-Cre-transgenic mice is infected with a MSCV-Stop-NPM-ALK-IRES-EGFP vector leading to expression of NPM-ALK in early T-cells. With a latency of 3-4 months, mice develop lymphomas and die from neoplastic T-cell-infiltration of BM and lymphatic organs. To investigate the impact of abrogation of CD30 signals on the development of NPM-ALK+ ALCL in our model, CD30 knockout mice were crossed with Lck-Cre mice. Both Lck-Cre NPM-ALK CD30 wt and Lck-Cre NPM-ALK CD30 ko recipients develop a human ALCL-like lymphoma with a pure T-cell phenotype characterized by Thy1.2+ cells infiltrating the thymus, lymph nodes, spleen and BM. First results from Lck-Cre NPM-ALK CD30 ko transplanted mice showed impaired disease induction and prolonged survival compared to CD30 wt animals. Moreover, secondary transplantation of NPM-ALK thymic lymphomas led to distinct deceleration of disease development upon CD30 deletion. Microarray analyses have shed some light on the mechanisms underlying the delayed lymphoma progression of CD30 deleted tumors with an upregulation of inflammatory pathways and proteins that are master players in inflammation and immune responses. Further characterization of the role of CD30-mediated immune response in disease progression using this mouse model and immunocompromised mice is ongoing. An improved understanding of how the immune system affects tumor progression will extend the rationale in translational strategies to use immunotherapies for patients with T-NHLs. Disclosures No relevant conflicts of interest to declare.

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.

Time- and tissue-specific expression of transgenes is essential for the accurate representation of human disease in in vivo models. To improve flexibility but also fidelity of ALK+ ALCL models, we developed new approaches enabling lineage specific expression of NPM-ALK cDNAs, as well as a novel system for CRISPR/Cas induced Npm-Alk recombination. First, we have designed a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop-cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is rapidly achieved through retroviral infection and subsequent transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage specific promoter. To validate the model, we directed expression of NPM-ALK, the fusion oncogene present in anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre transgenic mice with a retroviral construct containing the Npm-Alk cDNA preceded by a translational stop cassette. The approach efficiently induced T-cell lymphomas within 12-16 weeks closely resembling the human disease including the expression of the ALCL hallmark antigen CD30. Since NPM-ALK overexpressed from a cDNA acts as a very strong oncogene transforming a range of cell types, we were interested to develop a more physiologic model based on chromosomal recombination, enabling NPM-ALK expression from the endogenous Npm promoter. To achieve this, we have designed guide RNAs (gRNAs) directed to either the intron between exon 4 and 5 for Npm1 on mouse chr. 11, or the intron between exons 19 and 20 for Alk on mouse chr. 17., enabling targeted translocation between the two chromosomes. For further analysis, the IL-3 dependent murine pro-B cell line Ba/F3 stably expressing the Cas9 recombinase was transduced with the respective gRNAs and subsequently grown in the absence of IL-3 to allow positive selection of cells transformed by productive t(11;17) NA recombination. A PCR reaction on genomic DNA using primers covering the translocation breakpoint resulted in a product and Sanger sequencing of the amplicon confirmed the intended recombination at the targeted genomic positions. The translocation was also detectable by fluorescence in-situ hybridization (FISH), and Western blot analysis demonstrated expression of a highly phosphorylated Npm-Alk fusion protein. To further probe for oncogene dependency, we treated Npm-Alk translocated cells and control cells with a specific Alk inhibitor, resulting in rapid cell depletion of the Npm-Alk translocated cells, but not controls. The described Cre/loxP-based system represents a versatile tool for the rapid functional analysis of gene function in a defined lineage or in a developmental stage in vivo, and faithfully recapitulates many features of ALCL. Furthermore, using Crispr/Cas to induce targeted double-strand breaks, we have been able to generate specific Npm-Alk translocations in murine cells, paving the way for novel models which may help to further define the initial pathogenetic event underlying lymphomagenesis in ALCL. Disclosures No relevant conflicts of interest to declare.

Mutations in the colony-stimulating factor 3 receptor (CSF3R) have been identified in 90% of chronic neutrophilic leukemia (CNL) and lower frequency in atypical chronic myeloid leukemia (aCML). It was shown recently, that signaling of the different types of CSF3R mutations varies, but a central role of the JAK/STAT pathway activation by oncogenic CSF3R membrane proximal point mutations has been anticipated. However, the exact downstream signaling pathways mediated by CSF3R mutations are still not completely understood. Recent studies in JAK mutated myeloproliferative diseases uncovered the vital importance of JAK/STAT signaling in PD-L1 mediated immune escape, thereby suggesting a possible role and mechanistic link for PD-1/PD-L1 immune checkpoint regulation in CNL and aCML. To investigate the dependence of the oncogenic CSF3R signaling cascade on an activated JAK/STAT axis, we performed Ruxolitinib treatment in MTT assays with BaF3 cells retrovirally infected with pMIG-R1 vectors harboring either CSF3R point or truncation mutations. Our data indicate, that both, CSF3R point and truncation mutations induced cell growth could be inhibited by Ruxolitinib treatment. To narrow down the involvement of the different JAK family kinases, we included CSF3R mutation transduced JAK1 deficient U4C, JAK2 deficient y2A and TYK2 deficient U1A cell lines in combination with different JAK inhibitors. Interestingly, here we were able to show that CSF3R point mutations require functional JAK1/TYK2-STAT3 signaling for oncogenic potential, whereas truncation mutations depend on all JAK kinases through STAT3/5 for activation. To further study potential therapeutic strategies, we examined checkpoint inhibition in CSF3R mutated disease. In this regard we demonstrate a significant induction of PD-L1 expression in murine cell lines upon expression of CSF3R point mutations T615A or T618I. To investigate the impact of PD-L1 upregulation in vivo, we used a retroviral murine bone marrow (BM) transplantation model where we infected BM of Balb/c mice with a vector carrying CSF3R T618I point mutation and transplanted it into lethally irradiated recipients. Transplanted mice developed a CSF3R driven leukemia and died by leukocytosis and organ infiltration, whereas mice transplanted with BM harboring CSF3RT618I and additional ADA mutation failed to induce leukemia. To test checkpoint inhibition as a potential therapeutic strategy in vivo, CSF3RT618I transplanted mice were treated regularly with anti-PD-L1-antibody or isotype control. Interestingly, checkpoint inhibition led to reduced EGFP burden and prolonged survival of responder mice suggesting a treatment response of immunooncologic agents in these rare diseases. In immunophenotypic analyses of primary patient material, we were able to show increased PD-L1 expression in primary diseased myeloid cells isolated from peripheral blood of CNL and aCML patients, thereby defining this structure as a possible new therapeutic target. To summarize, our data expand the knowledge of oncogenic CSF3R signaling pathways and implicate an immunotherapeutic strategy by checkpoint inhibition in CSF3R driven CNL and atypical CML. Disclosures Lubbert: Teva: Other: Study drug; Celgene: Other: Travel Grant; Janssen: Honoraria, Research Funding.

Introduction: BCL6 is a key oncogene in lymphoma pathogenesis. Malignant lymphoid cells exploit several mechanisms to deregulate the expression of BCL6, including chromosomal translocations, somatic mutations in the promoter regulatory regions, or inactivation of the pathway that controls its degradation. FBXO11 was recently identified as a major ubiquitin ligase involved in the degradation of BCL6 and was found to be frequently inactivated by mutations or deletion in diffuse large B cell lymphoma (DLBCL). Thus, FBXO11 acts as an oncosuppressor in DLBCL by promoting the accumulation of BCL6. Given the prominent role of FBXO11 in regulating BCL6 stability, we searched for FBXO11 mutations in BCL6-positive lymphomas, other than DLBCL, and we investigated its role in lymphoma development in vivo. Methods: We sequenced the entire FBXO11 coding sequence by classical Sanger sequencing in 100 cases of follicular lymphoma (FL), 36 cases of Burkitt lymphoma (BL), 8 BL cell lines and 8 anaplastic large cell lymphoma cell lines, all BCL6-positive lymphomas. Moreover, we sequenced 50 cases of marginal zone B cell lymphoma (MZL), which show variable expression of BCL6. We functionally validated the FBXO11 mutations by developing mutant constructs and testing their ability to induce BCL6 and SNAIL degradation. We then applied the CRISPR/Cas9 system to disrupt the endogenous FBXO11 gene in BL cells and evaluated its effect on BCL6 stability. To dissect the in vivo role of FBXO11 in lymphomagenesis we generated conditional FBXO11 knock-out (KO) mice (FBXO11fl/fl) to delete protein expression in CD19-positive B cells. To investigate whether FBXO11 inactivation cooperates with c-myc in lymphomagenesis we crossed CD19/Cre-FBXO11fl/fl mice with Eμ-myc transgenic mice. Results: We identified FBXO11 mutations in BL cases and cell lines (10/44, 22.7%), one case of FL (1/100) and one case of MZL (1/50). In FL and MZL, the mutational analysis of tissue collected by microdissection showed that the mutation was specifically acquired by the high-grade component. The frequency of FBXO11 mutation in BL was further validated on an independent cohort of 51 BL cases (6/51, 11.7%), obtained from published data of a previous whole-exome sequencing study (Love et al. Nat Genet 2012). BL mutations were mostly missense mutations located in the functional CASH domains and also splice-site mutations that were never described before and that induced alternative splicing, as confirmed on the mRNA extracted from the tumor samples. All mutations produced a mutant FBXO11 with impaired ability to induce BCL6 and SNAIL degradation. CRISPR/Cas9 mediated KO of FBXO11 in BL cells resulted in an almost complete stabilization of BCL6, thus suggesting that FBXO11 is the main, if not unique, ubiquitin ligase that controls BCL6 stability in BL. Finally, we observed an acceleration of lymphoma development in the CD19/Cre-FBXO11fl/fl mice crossed with Eμ-myc transgenic mice. The lymphomas showed histologic features of high-grade disease with a more mature B-cell phenotype than the Eμ-myc tumors alone. Conclusions: Overall our results demonstrate that FBXO11 is frequently mutated in BL. All mutants identified impair the ability of FBXO11 to regulate the degradation of BCL6. Together with our previous findings (Duan et al. Nature 2012), this study shows that FBXO11 is mostly mutated in aggressive lymphomas such as DLBCL and BL, and suggests that FBXO11 mutations could contribute to their aggressiveness. In fact, we show that FBXO11 inactivation cooperates with c-myc in accelerating lymphomagenesis. We have established a novel murine lymphoma model that resembles more closely the human BL, providing a novel promising tool for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches with BCL6 and/or c-myc inhibitors. This abstract is also being presented as Poster 32. Citation Format: Chiara Pighi, Mara Compagno, Taek-Chin Cheong, Teresa Poggio, Qi Wang, Fernanda Langellotto, Anoop Sendamarai, Kyriacos Markianos, Paola Francia di Celle, Alberto Zamò, Roberto Chiarle. FBXO11 is recurrently mutated in Burkitt lymphoma and its inactivation accelerates lymphomagenesis in Eμ-myc mice [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR10.

Non–small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKI), but the treatment is successful for only a limited amount of time; most patients experience a relapse due to the development of drug resistance. Here, we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti–PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC. Cancer Immunol Res; 3(12); 1333–43. ©2015 AACR.

INTRODUCTION: We recently described inactivating mutations of the FBXO11 gene in Diffuse Large B Cell Lymphoma (DLBCL). One major function of FBXO11 is to regulate BCL6 stability as well as other targets such as SNAIL. BCL6 acts as an oncogene in several human B-cell lymphomas and its expression levels can be increased by several mechanisms including chromosomal translocations, point mutations in the promoter region and reduced degradation by inactivation of FBXO11. Thus, FBXO11 acts as an oncosuppressor in DLBCL by promoting the accumulation of BCL6. However, a clear and complete picture of the distribution of FBXO11 mutations in different lymphoma subtypes is missing, and the in vivo functions of FBXO11 in normal tissue and lymphoma development are completely lacking. In the present work, we investigated the frequency and distribution of FBXO11 mutations in an extended panel of human BCL6 positive lymphoma and we functionally validated novel FBXO11 mutations for their ability to induce BCL6 degradation. METHODS: We sequenced the entire FBXO11 coding sequence by classical Sanger sequencing in 100 cases of Follicular Lymphoma (FL), 36 cases of Burkitt Lymphoma (BL), 8 BL cell lines and 8 Anaplastic Large cell lymphoma (ALCL) cell lines, which are typically BCL6-positive lymphomas. Moreover, we sequenced 50 cases of Marginal Zone B cell Lymphoma (MZL), which show variable expression of BCL6. To investigate whether the FBXO11 mutations interfere with FBXO11 activity we generated complementary DNA constructs containing these mutations. Wild-type FBXO11 or FBXO11 mutants were expressed in HEK-293T cells with constructs expressing BCL6 or SNAIL. Twenty-four hours after transfection, HEK-293T cells were treated with cycloheximide and harvested at different time points to check substrates expression and degradation by immunoblotting. RESULTS: BL carried the highest frequency of FBXO11 mutations (12/44 cases analyzed, 27%), whereas FL and MZL were very rarely affected by FBXO11 mutations (1/100 FL and 1/50 MZL). The analysis identified several mutations, either located in the coding sequence mostly in the CASH (functional) domains or located in intronic sequences controlling exon splicing. Sequence changes included missense mutations (n=8), deletions (n=2), a frameshift deletion introducing a premature stop codon (n=1) and nucleotide substitutions at consensus splice donor sites (n=2) and acceptor site (n=1). Remarkably, these last mutations represent the first report of splice site mutations affecting the FBXO11 gene. By cDNA amplification and sequencing, we demonstrated skipping of entire exons: the splice site mutations located at the donor site of exon 15 and 19 lead to the loss of the exon 15 and 19 respectively, while the splice site mutation affecting the acceptor site of exon 16 results in the deletion of the adjacent exon 16. Two of the point mutations (K631N and Y122C), affecting one case of FL and MZL respectively, were also found in the previous panel of DLBCLs (Duan et al, Nature 2012). Both cases consisted of a low grade component adjacent to an area of high grade transformation. By microdissection, we separately studied the two components of each of these cases to demonstrate that the mutation was present only in the high grade component. Finally, to validate all the mutations found in BL, we generated mutant constructs corresponding to each of them. By this approach, we showed that all the mutations identified are loss of function variants because they impaired the FBXO11 ability to promote BCL6 and SNAIL degradation. CONCLUSIONS: We identified several FBXO11 novel mutations in a large panel of BCL6-positive human lymphomas. All the mutations identified were inactivating the FBXO11 ability to induce BCL6 and SNAIL degradation. In contrast, mutations of FBXO11 are rare in low grade FL and MZL and, when present, are associated with high grade transformation. Together with our previous findings, this study showed that FBXO11 is mostly mutated in aggressive lymphomas such as DLBCL and BL, thus suggesting that FBXO11 mutations could contribute to the de-novo onset or transformation into high grade lymphoma. Disclosures No relevant conflicts of interest to declare.

Activation-induced cytidine deaminase (AID) is a B cell-specific enzyme that initiates class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes, essential mechanisms to generate different classes of antibody and antibody diversity for the antigens. At lower frequency, AID also promiscuously introduces DNA structural lesions at non-Ig loci and it is involved in the pathogenesis of B cell lymphoma. Thus, its expression is tightly controlled in B cells to limit its genotoxic effects. Phosphatidylinositol 3-kinase (PI3K) p110δ isoform acts downstream of the B-Cell Receptor (BCR) to suppress AID expression, whereas blockade of PI3K signaling enhances the expression of AID. Potent oral PI3K inhibitors such as the p110δ inhibitor idelalisib (GS-1101, CAL-101) have been recently approved for the treatment of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and Waldenstrom macroglobulinemia (WM), whereas dual PI3K p110δ and γ inhibitors such as duvelisib (IPI-145) showed promising results for the treatment of CLL and other hematologic malignancies. Here we investigated whether the increased of AID expression caused by idelalisib or duvelisib induced genomic instability in normal and malignant B cells. We observed that in splenic purified mouse B cells activated with IL4 and αCD40 to induce AID expression and CSR, treatment with idelalisib and duvelisib significantly increased both AID protein and mRNA levels, compared to controls. As a result, both drugs strongly promoted CSR. The regulation of AID expression was strictly dependent on PI3K p110δ activity because a selective p110γ inhibitor (AS-604850) did not have any effect. In contrast, B cells expressing a constitutively active PI3Kδ showed suppression of CSR and reduced AID expression compared to inactive PI3Kδ. To gain insights into the degree of genomic instability induced by increased AID expression upon PI3Kδ inhibition, we applied a genome-wide translocation technique we previously developed (High-Throughput Genomic Translocation Sequencing approach, HTGTS) to identify translocation partners from DNA double strand breaks (DSBs) introduced into the c-myc locus (Chiarle et al, Cell 2011). By HTGTS, we isolated thousands of translocations from activated primary mouse B cells distributed widely across the genome. Remarkably, in B cells treated with idelalisib or duvelisib, we identified a significantly higher number of translocations in known AID off-target genes as well as novel hotspots of translocations (48 for idelalisib, 50 for duvelisib). Unbiased genome-wide analysis of translocation formation revealed a consistency in the translocation patterns, with AID target hotspots localized in the TSS region and predominantly grouped within super-enhancers and regulatory clusters. HTGTS analysis performed on activated AID knock-out (AID KO) B cells showed that the vast majority of these translocations were dependent on AID. Hence, our data demonstrate that in normal B cells PI3Kδ inhibitors increase genomic instability by an AID-dependent mechanism. Finally, we observed that both idelalisib and duvelisib increased AID expression in human EBV-immortalized and lymphoma cell lines (MCL and CLL). We adapted the HTGTS technique to human cells by introducing DSBs in the human myc locus by CRISPR/Cas9 technology and sequencing genome-wide the formation of chromosomal translocations. By this approach, we demonstrated that also in human neoplastic B cells the treatment with idelalisib or duvelisib increased the formation of translocations to known AID off targets. In conclusion, we showed that idelalisib or duvelisib increase genomic instability in normal and neoplastic B cells by enhancing AID expression. Since several B cell malignancies imply treatment with these drugs for years, these effects of PI3Kδ inhibitors on the genomic stability of B cells should be carefully taken into account for therapeutic outcomes and protocol design. Disclosures No relevant conflicts of interest to declare.

Most of Anaplastic Large Cell Lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein nucleophosmin (NPM)-ALK. NPM provides an oligomerization domain that causes the spontaneous dimerization and ligand-independent activation of the ALK. NPM-ALK deregulated kinase activity drives several pathways involved in cellular proliferation and survival and sustains the process of malignant transformation. NPM-ALK is localized both in the cytoplasm and the nucleus, but the role of NPM-ALK cytoplasmic or nuclear fractions on ALCL phenotype maintenance is undetermined. We found that in all ALK+ ALCL cell lines and in primary ALCL NPM-ALK was equally distributed between cytoplasm and nucleus, but only the cytoplasmic portion, formed by NPM-ALK homodimers, was kinetically active and had transforming properties. The nuclear portion, formed by NPM/NPM-ALK heterodimers, was inactive. Thus, about 50% of the NPM-ALK fusion is sequestered and inactivated in the nucleus of ALCL cells by WT NPM1. Indeed, relocalization of NPM-ALK entirely to the cytoplasm in NPM-/- cells or its overexpression in several ALK+ ALCL cell lines resulted in cell death through activation of the ERK1/2 pathway and of γ-H2AX, a key player of the DNA damage response pathway. Thus, excessive oncogenic ALK signaling induces a cell stress leading to apoptosis and a balanced amount of ALK activation is necessary for optimal ALCL growth. Interestingly, we selected three human NPM-ALK positive cell lines resistant to the dual ALK/EGFR inhibitor AP26113. Resistance was due to genomic NPM-ALK amplification that caused overexpression of NPM-ALK and consequently hyperactivation of downstream ALK-dependent pathways. These cell lines were not only resistant, but also drug-dependent, meaning that addition of a low amount of AP26113 was required for cellular growth and to keep “normal” NPM-ALK signalling levels. Indeed, upon drug withdrawal, NPM-ALK and its downstream pathways were hyperactivated. The viability of all three drug-addicted cell lines dramatically dropped, confirming that an excess of NPM-ALK signaling was detrimental for cell growth. In two resistant cell lines out of three a new population recovered in the absence of the drug and was re-sensitized to AP26113. Notably, in these new drug-sensitive populations NPM-ALK overexpression was abrogated. All together, these findings support the idea that deregulation of NPM-ALK localization might be an attractive therapeutic option. Moreover, for a subset of patients that may develop drug dependency as well as drug resistance as a consequence of NPM-ALK overexpression, a discontinuous tyrosine kinase inhibitor therapeutic schedule may be more effective than a continuous one, as it is usually proposed. Citation Format: Monica Ceccon, Maria Elena Boggio Merlo, Luca Mologni, Lydia Varesio, Teresa Poggio, Matteo Menotti, Silvia Bombelli, Roberta Rigolio, Andrea Manazza, Chiara Ambrogio, Giovanni Giudici, Cesare Casati, Mara Compagno, Suzanne Turner, Carlo Gambacorti Passerini, Roberto Chiarle, Claudia Voena. Excess of cytoplasmic NPM-ALK driven oncogenic signaling is toxic and promotes cellular apoptosis and drug dependency. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2015-2920

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. More frequently ALK is juxtaposed to the echinoderm microtubule-associated protein-like 4 (EML4) gene on chromosome 2 and generates a constitutively active EML4-ALK fusion protein that triggers downstream oncogenic signals leading to increased cell proliferation and survival. The majority of ALK-rearranged NSCLC presents a peculiar histology characterized by a solid signet-ring cell and a mucinous cribriform pattern that is frequently associated with a metastatic phenotype. As the signet ring phenotype and metastasis are associated with epithelial-mesenchymal transition (EMT), a cellular reprogramming often activated in cancer cells during invasion and metastasis, we investigated whether ALK induces EMT in NSCLC. We performed RNA sequencing analysis on human ALK-rearranged NSCLC cell lines treated with ALK inhibitors or where EML4-ALK was knocked-down by shRNA. We found that EML-ALK regulated several genes related to EMT. In particular, the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2), key regulators of a splicing switch during EMT, were repressed in ALK-rearranged NSCLC cells and their repression was dependent on ALK activity. In keeping with these observations, H2228 and DFCI032 cells displayed a mesenchymal phenotype with almost complete suppression of the epithelial marker, E-cadherin, and a strong expression of the mesenchymal markers, vimentin and N-cadherin. In H2228 and DFCI032 cells, both E-cadherin suppression and vimentin up-regulation were dependent upon EML-ALK kinase activity because treatment with ALK inhibitors (TAE684 and crizotinib) or ALK knock-down reverted the phenotype of H2228 and DFCI032 from mesenchymal to epithelial and decreased their invasive potential. We excluded the involvement in EMT of ALK-rearranged NSCLC of other RTKs, such as EGFR or MET, because their inhibition did not have any effect on E-cadherin and vimentin expression. In ALK-rearranged NSCLC, the regulation of E-cadherin suppression was mainly transcriptional whereas vimentin regulation was post-transcriptional for both cell lines. Overexpression of ESRP1 led to up-regulation of E-cadherin, whereas ESRP1 knock-down impaired the reversion to an epithelial phenotype associated to inhibition of ALK activity. In conclusion, we showed that oncogenic ALK regulates EMT in NSCLC through ESRP repression. These findings could have implications for the biology of ALK-rearranged NSCLC in terms of metastatic potential and resistance to therapy. Citation Format: Claudia Voena, Lydia Varesio, Liye Zhang, Matteo Menotti, Teresa Poggio, Filomena Di Giacomo, Elena Panizza, Cristina Mastini, Mara Compagno, Stefano Monti, Roberto Chiarle. ALK oncogene regulates epithelial-mesenchymal transition (EMT) in ALK-rearranged non-small cell lung carcinoma through repression of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 131. doi:10.1158/1538-7445.AM2015-131

T cell non-Hodgkin lymphoma (T-NHL) is a rare and heterogeneous group of neoplasms of the lymphoid system. With the exception of a few relatively indolent entities, T-NHL is typically aggressive, treatment resistant, and associated with poor prognosis. Relatively few options with proven clinical benefit are available for patients with relapsed or refractory disease. Immunotherapy has emerged as a promising treatment for the management of patients with hematological malignancies. The identification of tumor antigens has provided a large number of potential targets. Therefore, several monoclonal antibodies (alemtuzumab, SGN-30, brentuximab vedotin, and mogamulizumab), directed against tumor antigens, have been investigated in different subtypes of T-NHL. In addition to targeting antigens involved in cancer cell physiology, antibodies can stimulate immune effector functions or counteract immunosuppressive mechanisms. Chimeric antigen receptor (CAR)-T cells directed against CD30 and immune checkpoint inhibitors are currently being investigated in clinical trials. In this review, we summarize the currently available clinical evidence for immunotherapy in T-NHL, focusing on the results of clinical trials using first generation monoclonal antibodies, new immunotherapeutic agents, immune checkpoint inhibitors, and CAR-T cell therapies.