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Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig­naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC. Significance: HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease. This article is highlighted in the In This Issue feature, p. 1825

Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig­naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC.Significance:HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Data from IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma

Genetic analyses of BRCA1 mutant cell lines and PDX models.

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo. Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778–90. ©2016 AACR.

Legend for Supplementary Figures S1-S6.

RNA-seq analyses of global exon splicing events (S1); Cell line therapy sensitivities (S2); Hazard ratios for survival of patients with serous ovarian carcinomas associated with BRCA1 mutation, age at diagnosis and stage (S3); Characteristics of studies included in survival analysis from Fig. 5A (S4).

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Supplementary Figure 3 from A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

This file contains supplementary figures 1-4. Supplementary Figure 1 provides supporting information for the effects of Rpl22 loss on thymic cellularity and supports Figure 1. Supplementary Figure 2 describes the phenotypes of periperhal T lineage cells, both normal and transformed, in Rpl22-sufficient and Rpl22-deficient mice. It supports the data in Figure 3. Supplementary Figure 3 depicts the maturation of single positive thymocytes in MyrAkt Tg Rpl22-deficient mice, revealing that maturation is not impaired. These data support Figure 5. Supplementary figure 4 depicts the effect of Rpl22 mutation on expression of KLF2 and S1PR1 in human T-ALL. It supports the data in Figure 6.

Supplementary Figure 4 from A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

The oncogene v-akt was isolated from a retrovirus that induced murine thymic lymphomas. Transgenic mice expressing a constitutively activated form of the cellular homologue Akt2 specifically in immature T cells develop spontaneous thymic lymphomas. We hypothesized that tumors from these mice might exhibit oncogenic chromosomal rearrangements that cooperate with activated Akt2 in lymphomagenesis. Cytogenetic analysis revealed a recurrent clonal inversion of chromosome 6, inv(6), in thymic lymphomas from multiple transgenic founder lines, including one line in which 15 of 15 primary tumors exhibited this same rearrangement. Combined fluorescence in situ hybridization, PCR, and DNA sequence analyses showed that the distal inv(6) breakpoint resides at the T-cell receptor β chain locus, Tcrb. The proximal breakpoint maps to a region near a locus comprising the linked homeobox/transcription factor genes Dlx5 and Dlx6. Expression analysis of genes translocated to the vicinity of the Tcrb enhancer revealed that Dlx5 and Dlx6 are overexpressed in tumors exhibiting the inv(6). Experimental overexpression of Dlx5 in mammalian cells resulted in enhanced cell proliferation and increased colony formation, and clonogenic assays revealed cooperativity when both Dlx5 and activated Akt2 were coexpressed. In addition, DLX5, but not DLX6, was found to be abundantly expressed in three of seven human T-cell lymphomas tested. These findings suggest that the Dlx5 can act as an oncogene by cooperating with Akt2 to promote lymphomagenesis. [Cancer Res 2008;68(5):1296–302]

Mutations in ribosomal proteins cause bone marrow failure syndromes associated with increased cancer risk, but the basis by which they do so remains unclear. We reported previously that the ribosomal protein Rpl22 is a tumor suppressor in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), and that loss of just one Rpl22 allele accelerates T-cell lymphomagenesis by activating NF-κB and inducing the stem cell factor Lin28B. Here, we show that, paradoxically, loss of both alleles of Rpl22 restricts lymphoma progression through a distinct effect on migration of malignant cells out of the thymus. Lymphoma-prone AKT2-transgenic or PTEN-deficient mice on an Rpl22−/− background developed significantly larger and markedly more vascularized thymic tumors than those observed in Rpl22+/+ control mice. But, unlike Rpl22+/+ or Rpl22+/− tumors, Rpl22−/− lymphomas did not disseminate to the periphery and were retained in the thymus. We traced the defect in the Rpl22−/− lymphoma migratory capacity to downregulation of the KLF2 transcription factor and its targets, including the key migratory factor sphingosine 1-phosphate receptor 1 (S1PR1). Indeed, reexpression of S1PR1 in Rpl22-deficient tumor cells restores their migratory capacity in vitro. The regulation of KLF2 and S1PR1 by Rpl22 appears to be proximal as Rpl22 reexpression in Rpl22-deficient lymphoma cells restores expression of KLF2 and S1P1R, while Rpl22 knockdown in Rpl22-sufficient lymphomas attenuates their expression. Collectively, these data reveal that, while loss of one copy of Rpl22 promotes lymphomagenesis and disseminated disease, loss of both copies impairs responsiveness to migratory cues and restricts malignant cells to the thymus. Cancer Res; 76(11); 3387–96. ©2016 AACR.

This file contains the legends fro supplementary figures 1-4.

Supplementary Figure 2 from A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

Supplementary Figure 1 from A Novel Recurrent Chromosomal Inversion Implicates the Homeobox Gene Dlx5 in T-Cell Lymphomas from Lck-Akt2 Transgenic Mice

While the functions of αβ T cells in host resistance to pathogen infection are understood in far more detail than those of γδ lineage T cells, γδ T cells perform critical, essential functions during immune responses that cannot be compensated for by αβ T cells. Accordingly, it is critical to understand how the development of γδ T cells is controlled so that their generation and function might be manipulated in future for therapeutic benefit. This introductory chapter will focus primarily on the basic processes that underlie γδ T cell development in the thymus, as well as the current understanding of how they are controlled.

While Ras/mitogen-activated protein kinase (MAPK) signaling is activated in most human cancers, attempts to target this pathway using kinase active site inhibitors have not typically led to durable, clinical benefit. To address this shortcoming, we sought to test the feasibility of an alternative targeting strategy, focused on the ERK2 substrate binding domains, D and DBP. We found that disabling the ERK2-DBP domain in mice caused baseline erythrocytosis. Consequently, we investigated the role of the ERK2-D and -DBP domains in disease, using a JAK2-dependent model of polycythemia vera (PV). Importantly, inactivation of the ERK2-DBP domain promoted the progression of disease from PV to myelofibrosis (MF), suggesting that the ERK2-DBP domain normally opposes progression. ERK2-DBP inactivation also prevented oncogenic JAK2 kinase (JAK2V617F) from promoting oncogene-induced senescence (OIS) in vitro. The ERK2-DBP mutation attenuated JAK2-mediated OIS by preventing the physical interaction of ERK2 with the transcription factor, Egr1. Because inactivation of the ERK2-DBP created a functional ERK2 kinase limited to binding substrates through its D-domain, these data suggested that the D-domain substrates were responsible for promoting oncogene-induced progenitor growth and tumor progression, and that pharmacologic targeting of the ERK2-D domain might attenuate cancer cell growth. Indeed, pharmacologic agents targeting the ERK2-D domain were effective in attenuating the growth of JAK2-dependent myeloproliferative neoplasm cell lines. Taken together, these data indicate that the ERK-D and -DBP domains can play distinct roles in the progression of neoplasms and that the D-domain has the potential to be potent therapeutic target in Ras/MAPK dependent cancers.

Ribosomal proteins are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein. However, in this study, we show that Rpl22-like1 (Rpl22l1) regulates hematopoiesis without affecting ribosome biogenesis or bulk protein synthesis. Conditional loss of murine Rpl22l1 using stage or lineage-restricted Cre drivers impairs development of several hematopoietic lineages. Specifically, Tie2-Cre–mediated ablation of Rpl22l1 in hemogenic endothelium impairs the emergence of embryonic hematopoietic stem cells. Ablation of Rpl22l1 in late fetal liver progenitors impairs the development of B lineage progenitors at the pre-B stage and development of T cells at the CD44−CD25+ double-negative stage. In vivo labeling with O-propargyl-puromycin revealed that protein synthesis at the stages of arrest was not altered, indicating that the ribosome biogenesis and function were not generally compromised. The developmental arrest was associated with p53 activation, suggesting that the arrest may be p53-dependent. Indeed, development of both B and T lymphocytes was rescued by p53 deficiency. p53 induction was not accompanied by DNA damage as indicated by phospho-γH2AX induction or endoplasmic reticulum stress, as measured by phosphorylation of EIF2α, thereby excluding the known likely p53 inducers as causal. Finally, the developmental arrest of T cells was not rescued by elimination of the Rpl22l1 paralog, Rpl22, as we had previously found for the emergence of hematopoietic stem cells. This indicates that Rpl22 and Rpl22l1 play distinct and essential roles in supporting B and T cell development.

SUMMARY TCF1 plays a critical role in T lineage commitment and the development of αβ lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis., Graphical Abstract, In Brief Fahl et al. reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element, and this regulatory axis modulates γδ lineage commitment and effector fate. This finding provides mechanistic insight into how TCR signals promote adoption of the γδ17 effector fate.

γδ T cells are a subset of T cells with attributes of both the innate and adaptive arms of the immune system. These cells have long been an enigmatic and poorly understood component of the immune system and many have viewed them as having limited importance in host defense. This perspective persisted for some time both because of critical gaps in knowledge regarding how the development of γδ T cells is regulated and because of the lack of effective and sophisticated approaches through which the function of γδ T cells can be manipulated. Here, we discuss the recent advances in both of these areas, which have brought the importance of γδ T cells in both productive and pathologic immune function more sharply into focus.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset—accessible through the Beat AML data viewer (Vizome)—that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML. Analyses of samples from patients with acute myeloid leukaemia reveal that drug response is associated with mutational status and gene expression; the generated dataset provides a basis for future clinical and functional studies of this disease.

γδ T‐cells perform a wide range of tissue‐ and disease‐specific functions that are dependent on the effector cytokines produced by these cells. However, the aggregate signals required for the development of interferon‐γ (IFNγ) and interleukin‐17 (IL‐17) producing γδ T‐cells remain unknown. Here, we define the cues involved in the functional programming of γδ T‐cells, by examining the roles of T‐cell receptor (TCR), Notch, and cytokine‐receptor signaling. KN6 γδTCR‐transduced Rag2 −/− T‐cell progenitors were cultured on stromal cells variably expressing TCR and Notch ligands, supplemented with different cytokines. We found that distinct combinations of these signals are required to program IFNγ versus IL‐17 producing γδ T‐cell subsets, with Notch and weak TCR ligands optimally enabling development of γδ17 cells in the presence of IL‐1β, IL‐21 and IL‐23. Notably, these cytokines were also shown to be required for the intrathymic development of γδ17 cells. Together, this work provides a framework of how signals downstream of TCR, Notch and cytokine receptors integrate to program the effector function of IFNγ and IL‐17 producing γδ T‐cell subsets.

Unlike αβ-T lineage cells, where the role of ligand in intrathymic selection is well established, the role of ligand in the development of γδ-T cells remains controversial. Here we provide evidence for the role of a bona fide selecting ligand in shaping the γδ-T cell-receptor (TCR) repertoire. Reactivity of the γδ-TCR with the major histocompatibility complex (MHC) Class Ib ligands, H2-T10/22, is critically dependent upon the EGYEL motif in the complementarity determining region 3 (CDR3) of TCRδ. In the absence of H2-T10/22 ligand, the commitment of H2-T10/22 reactive γδ-T cells to the γδ fate is diminished, and the specification of those γδ committed cells to the IFN-γ or interleukin-17 effector fate is altered. Furthermore, those cells that do adopt the γδ fate and mature exhibit a profound alteration in the γδTCR repertoire, including depletion of the EGYEL motif and reductions in both CDR3δ length and charge. Taken together, these data suggest that ligand plays an important role in shaping the TCR repertoire of γδ-T cells.

The interaction between ERK2 and its substrates is critically mediated by two domains, the common docking (CD) D-domain and DEF-binding pocket (DBP) domain. Previous studies have suggested that ERK2 is not only necessary to drive hematopoietic and myelo-erythroid development, but it is also important for the pathogenesis of hematological cancers, as revealed by recurrent ERK2 somatic mutations in many types of lymphoma and leukemia. Here we show that the activation of ERK2 in JAK2V617F-driven myeloproliferative neoplasm (MPN) enhance polycythemia vera (PV) progression from erythrocytosis to myelofibrosis when ERK2-DBP binding capacity is disabled. Conversely, targeting the ERK2 D-domain preserves ERK2 catalytic function while reducing outgrowth and proliferation of human and murine MPN cell lines. To determine whether ERK2 binding domains play a role in the pathogenesis of JAK2V617F-driven MPN, we generated an ERK2-Y261A (Erk2 Y261A) mutant knockin mouse model in which the ERK2-DBP domain was inactivated, but ERK2 kinase activity and D-domain function was preserved. We observed splenomegaly in Erk2 Y261A/Y261A, characterized by a 10-fold expansion of CD44+/Ter119+ primitive erythroblasts and Ter119+CD44+/ FSC hi immature erythroid progenitors. We then transplanted Jak2 V617F-expressing ERK2 wildtype (WT), knockout (KO) or DBP hematopoietic progenitors (HSPCs) into irradiated, immunodeficient mice (Rag2 −/−Il2rg −/−) and observed disease resembling human PV, with elevated RBC counts, hematocrit, and hemoglobin levels. ERK2-DBP mutant recipients exhibited mild but sustained erythrocytosis with a prevalence of circulating GFP+ monocytes and neutrophils. At 12-weeks post-transplantation, ERK2-DBP mutant recipients had significant splenic burden and decreased bone marrow cellularity relative to WT and KO recipients. Reticulin staining confirmed progression towards myelofibrosis with interstitial infiltration in both the BM and the spleen of ERK2 DBP mutant recipients. To understand the molecular basis of ERK-DBP domain in JAK2V617F-driven MPN, we performed in vitro colony forming unit assay to assess the clonogenic potential of JAK2V617F-expressing HSPCs in cytokine supplemented-methylcellulose supporting myeloid or erythroid progenitor development. Ectopic JAK2V617F expression markedly reduced colony formation by both ERK2 wildtype and knockout HSPCs. On the other hand, JAK2V617F expression did not reduce colony formation by ERK2-DBP HSPCs. These differences were not evident in methylcellulose cultures that supported erythroid development, suggesting that disabling ERK-DBP binding capacity primarily affects the myeloid lineage. Furthermore, we found that JAK2V617F increased senescence associated β-galactosidase (SA-βGa) activity in ERK2-WT HSPCs. SA-βGal activity was modestly affected by ERK deficiency but was strongly attenuated by the ERK2-DBP mutation, supporting the notion that ERK2-DBP domain promotes oncogene-induced senescence (OIS). Previously, we identified transcription factor, Early growth response 1 (Egr1) which interacts with ERK2-DBP domain and plays a critical role as a tumor suppressor in myeloid neoplasms. We observed that JAK2V617F expression failed to suppress colony formation and senescence by Egr1-deficient HSPCs. The ability of EGR1 to restore these effects is abrogated by inactivating the DEF motif (EGR1-Y252A). Together, this data demonstrates that the interaction between ERK2-DBP and Egr1 is required for JAK2V617F-mediated colony suppression and senescence induction. Finally, to determine whether ERK-D domain acts to promote progression by targeting substrates distinct from those that bind to the DBP domain, we developed an inhibitor (#76) of the ERK-D domain. Indeed, 76 spared ERK phosphorylation and EGR1 induction but impaired RSK phosphorylation, a critical D-domain substrate involved in cancer progression. Moreover, 76 suppressed colony formation by JAK2V61F-expressing HSPCs akin to the active site MEK/ERK inhibitor, U0126. By altering the 4-ethoxybenylidene moiety of 76, we were able to enhance anti-proliferative effects of the parent compound against human MPN cell lines, SET-2, UKE-1, and Ku812. Our findings identify ERK2-domain specific roles in the pathogenesis of JAK2V617F driven MPN and supports a novel therapeutic approach to targeting JAK2 and MAPK dependent MPN. Disclosures Levine: Celgene: Research Funding; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Lilly: Honoraria; Janssen: Consultancy; Astellas: Consultancy; Gilead: Honoraria; Prelude: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy; Roche: Honoraria, Research Funding; Imago: Membership on an entity's Board of Directors or advisory committees. Wiest: Janssen Pharmaceuticals: Research Funding.

Introduction: While most patients (pts) with cutaneous T-cell lymphoma (CTCL) have an indolent course, survival for stages ≥IIB is usually less than 5 years (Kim YH et al, Arch Dermatol 2003). When an aggressive approach of combination cytotoxic therapies and radiation in CTCL was compared to conservative sequential therapies in newly diagnosed pts, the increase in response rate with combination therapy was offset by toxicities, and no benefit in disease-free or overall survival was seen (Kaye FS et al, NEJM, 1989). Since then several novel agents have been approved in CTCL, including the CD30-directed antibody-drug conjugate brentuximab vedotin (BV) [in relapsed primary cutaneous ALCL and CD30-expressing mycosis fungoides (MF)], and HDAC inhibitors (HDACi). However, response rates of single agents are modest. Tolerable and more efficacious therapies are needed, including rational combinations of active biological agents. Evidence suggests that HDAC inhibition may upregulate CD30 expression (Hasanali ZS et al, Sci Transl Med 2015), supporting the combination of the HDACi romidepsin (R) with Brentuximab vedotin (BV) in pts with CTCL. Methods: In this multicenter phase I clinical trial, pts age ≥18 with stage ≥IB CTCL, good organ function, ECOG PS≤2, Results: At the time of abstract submission, 7 pts have been enrolled (DL0: n=3; DL1: n=3; expansion cohort: n=1). No pt experienced a cycle 1 DLT and DL1 was deemed the MTD. No pts were enrolled in the de-escalation cohorts. Enrollment in the expansion cohort is ongoing. Median age of pts was 64 years (range 51-79); 72% (n=5) were male; median ECOG PS 1 (0-2); median prior lines of systemic therapy were 4 (0-4), including 1 pt with prior HDACi, and 1 pt with prior BV and R exposure. All pts had MF. Stage at enrollment was stage IIB in 5 (72%), IB and IVA2 in 1 each (14% each). No pts have experienced G4 or 5 adverse events (AE). The only G3 AEs observed during treatment were transaminitis and fever (n=1 each), both resolved spontaneously. The most common AEs were nausea (71%), vomiting (43%), gastro-esophageal reflux, constipation, peripheral sensory neuropathy, anorexia, fatigue, and thrombophlebitis (29% each; see Table 1). Response assessment is available for 5 of 7 pts. The overall response rate was 80% (4/5), all of which partial responses, including 1 pt who had received both prior R and BV. The median change in mSWAT was a decrease of 59% from baseline (range -19.5 to -81.8%). After a median follow up of 6.1 months, median estimated progression-free survival was 12 months (PFS probability 0.42; 95%CI 0.1-1.0). Four pts came off treatment: 2 due to progression, 1 due to non-adherence related to COVID-19 concerns, 1 because of recurrent thrombophlebitis; 3 pts remain on treatment. Conclusion: Preliminary findings from this phase I study exploring the combination of R+BV indicate that R+BV is well tolerated at a dose of R 14mg/m2 and BV 1.2mg/kg given every 2 weeks and appears efficacious in CTCL. Updated results will be presented at the time of the meeting. Enrollment in the expansion cohort and correlative studies, including analysis of changes in CD30 expression after 1 dose of R, and association of response with CD30 expression, are ongoing. Disclosures Barta: Atara: Honoraria; Monsanto: Consultancy; Seattle Genetics: Honoraria, Research Funding; Janssen: Honoraria; Pfizer: Honoraria. Feldman:AstraZeneca: Consultancy; Janssen: Speakers Bureau; Portola: Research Funding; Pfizer: Research Funding; Kyowa Kirin: Consultancy, Research Funding; Eisai: Research Funding; Cell Medica: Research Funding; Amgen: Research Funding; Pharmacyclics: Honoraria, Other, Speakers Bureau; Abbvie: Honoraria; Bayer: Consultancy, Honoraria; Trillium: Research Funding; Viracta: Research Funding; Rhizen: Research Funding; Corvus: Research Funding; BMS: Consultancy, Honoraria, Research Funding; Kite: Honoraria, Other: Travel expenses, Speakers Bureau; Celgene: Honoraria, Research Funding; Takeda: Honoraria, Other: Travel expenses; Seattle Genetics, Inc.: Consultancy, Honoraria, Other: Travel expenses, Research Funding, Speakers Bureau. DeSimone:Sanofi/Genzyme: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Soligenix: Other: Investigator; Helsinn: Speakers Bureau. Fung:Genentech: Honoraria, Other: speakers' bureau, travel support; Sanotif: Honoraria, Other: speakers' bureau, travel support; AstraZeneca: Honoraria, Other: speakers' bureau, travel support; Kite, a Gilead Company: Honoraria, Other: speakers' bureau, travel support; Takeda: Honoraria, Other: speakers' bureau, travel support; Janssen Oncology: Honoraria, Other: speakers' bureau, travel support; AbbVie: Honoraria, Other: speakers' bureau, travel support. Khan:Celgene: Research Funding; Seattle Genetics: Research Funding; Janssen: Honoraria; Pharmacyclics: Honoraria; Bristol Myers Squibb: Research Funding. OffLabel Disclosure: Combination of romidepsin and brentuximab vedotin.

SUMMARY γδ T cells form an integral arm of the immune system and are critical during protective and destructive immunity. However, how γδ T cells are functionally programmed in vivo remains unclear. Here, we employ RBPJ-inducible and KN6-transgenic mice to assess the roles of ontogenic timing, T cell receptor (TCR) signal strength, and Notch signaling. We find skewing of Vγ1+ cells toward the PLZF+Lin28b+ lineage at the fetal stage. Generation of interleukin-17 (IL-17)-producing γδ T cells is favored during, although not exclusive to, the fetal stage. Surprisingly, Notch signaling is dispensable for peripheral γδ T cell IL-17 production. Strong TCR signals, together with Notch, promote IL-4 differentiation. Conversely, less strong TCR signals promote Notch-independent IL-17 differentiation. Single-cell transcriptomic analysis reveals differential programming instilled by TCR signal strength and Notch for specific subsets. Thus, our results precisely define the roles of ontogenic timing, TCR signal strength, and Notch signaling in γδ T cell functional programming in vivo., In brief Ontogeny and TCR signal strength are known to influence γδ T cell differentiation. Chen et al. show that temporal control of Notch signaling in RBPJ-inducible mice affects innate γδ T cell differentiation. TCR signal strength and Notch influence IL-4 versus IL-17 γδ T cell programming, which correlate with low versus high Ccr9 expression, respectively., Graphical Abstract

Summary: Most ribosomal proteins (RP) are regarded as essential, static components that contribute only to ribosome biogenesis and protein synthesis. However, emerging evidence suggests that RNA-binding RP are dynamic and can influence cellular processes by performing “extraribosomal,” regulatory functions involving binding to select critical target mRNAs. We report here that the RP, Rpl22, and its highly homologous paralog Rpl22-Like1 (Rpl22l1 or Like1) play critical, extraribosomal roles in embryogenesis. Indeed, they antagonistically control morphogenesis through developmentally regulated localization to the nucleus, where they modulate splicing of the pre-mRNA encoding smad2, an essential transcriptional effector of Nodal/TGF-β signaling. During gastrulation, Rpl22 binds to intronic sequences of smad2 pre-mRNA and induces exon 9 skipping in cooperation with hnRNP-A1. This action is opposed by its paralog, Like1, which promotes exon 9 inclusion in the mature transcript. The nuclear roles of these RP in controlling morphogenesis represent a fundamentally different and paradigm-shifting mode of action for RP. : Zhang et al. reveal that ribosomal proteins can perform regulatory functions of fundamental importance while separated from the ribosome. They demonstrate that ribosomal proteins Rpl22 and Rpl22-Like1 (Like1) are retained in the nucleus during gastrulation, where they control morphogenesis by modulating the splicing of Smad2 pre-mRNA in cooperation with HNRNP-A1. Keywords: ribosomal protein, paralog, morphogenesis, gastrulation, extraribosomal function, Rpl22, Rpl22l1, pre-mRNA splicing, Smad2, hnRNP-A1

// Yinfei Tan 1, * , Eleonora Sementino 1, * , Jinfei Xu 1 , Jianming Pei 1 , Zemin Liu 1 , Timothy K. Ito 1 , Kathy Q. Cai 2 , Suraj Peri 3 , Andres J.P. Klein-Szanto 1, 2 , David L. Wiest 4 , Joseph R. Testa 1 1 Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA 2 Histopathology Facility, Fox Chase Cancer Center, Philadelphia, PA 19111, USA 3 Department of Biostatistics and Bioinformatics, Fox Chase Cancer Center, Philadelphia, PA 19111, USA 4 Blood Cell Development and Function Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA * These authors contributed equally to this work Correspondence to: Joseph R. Testa, email: Joseph.Testa@fccc.edu Keywords: T cell lymphoma, Notch, Akt, Dlx5, homeobox Received: November 09, 2016 Accepted: January 11, 2017 Published: January 21, 2017 ABSTRACT Homeobox genes play a critical role in embryonic development, but they have also been implicated in cancer through mechanisms that are largely unknown. While not expressed during normal T-cell development, homeobox transcription factor genes can be reactivated via recurrent chromosomal rearrangements in human T-cell acute leukemia/lymphoma (T-ALL), a malignancy often associated with activated Notch and Akt signaling. To address how epigenetic reprogramming via an activated homeobox gene might contribute to T-lymphomagenesis, we investigated a transgenic mouse model with thymocyte-specific overexpression of the Dlx5 homeobox gene. We demonstrate for the first time that Dlx5 induces T-cell lymphomas with high penetrance. Integrated ChIP-seq and mRNA microarray analyses identified Notch1/3 and Irs2 as direct transcriptional targets of Dlx5, a gene signature unique to lymphomas from Lck-Dlx5 mice as compared to T-cell lymphomas from Lck-MyrAkt2 mice, which were previously reported by our group. Moreover, promoter/enhancer studies confirmed that Dlx5 directly transactivates Notch expression. Notch1/3 expression and Irs2-induced Akt signaling were upregulated throughout early stages of T-cell development, which promoted cell survival during β-selection of T lymphocytes. Dlx5 was required for tumor maintenance via its activation of Notch and Akt, as tumor cells were highly sensitive to Notch and Akt inhibitors. Together, these findings provide unbiased genetic and mechanistic evidence that Dlx5 acts as an oncogene when aberrantly expressed in T cells, and that it is a novel discovery that Notch is a direct target of Dlx5. These experimental findings provide mechanistic insights about how reactivation of the Dlx5 gene can drive T-ALL by aberrant epigenetic reprogramming of the T-cell genome.

The transcription factor JUN is frequently overexpressed in multiple genetic subtypes of acute myeloid leukemia (AML); however, the functional role of JUN in AML is not well defined. Here we report that short hairpin RNA (shRNA)-mediated inhibition of JUN decreases AML cell survival and propagation in vivo. By performing RNA sequencing analysis, we discovered that JUN inhibition reduces the transcriptional output of the unfolded protein response (UPR), an intracellular signaling transduction network activated by endoplasmic reticulum (ER) stress. Specifically, we found that JUN is activated by MEK signaling in response to ER stress, and that JUN binds to the promoters of several key UPR effectors, such as XBP1 and ATF4, to activate their transcription and allow AML cells to properly negotiate ER stress. In addition, we observed that shRNA-mediated inhibition of XBP1 or ATF4 induces AML cell apoptosis and significantly extends disease latency in vivo tying the reduced survival mediated by JUN inhibition to the loss of pro-survival UPR signaling. These data uncover a previously unrecognized role of JUN as a regulator of the UPR as well as provide key new insights into the how ER stress responses contribute to AML and identify JUN and the UPR as promising therapeutic targets in this disease.

Although ribosomal proteins (RP) are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein, emerging evidence suggests that individual RP can perform critical regulatory functions that control developmental processes. We showed previously that despite the ubiquitous expression of the RP ribosomal protein L22 (Rpl22), germline ablation of Rpl22 in mice causes a selective, p53-dependent block in the development of αβ, but not γδ, T cell progenitors. Nevertheless, the basis by which Rpl22 loss selectively induces p53 in αβ T cell progenitors remained unclear. We show in this study that Rpl22 regulates the development of αβ T cells by restraining endoplasmic reticulum (ER) stress responses. In the absence of Rpl22, ER stress is exacerbated in αβ, but not γδ, T cell progenitors. The exacerbated ER stress in Rpl22-deficient αβ T lineage progenitors is responsible for selective induction of p53 and their arrest, as pharmacological induction of stress is sufficient to induce p53 and replicate the selective block of αβ T cells, and attenuation of ER stress signaling by knockdown of protein kinase R–like ER kinase, an ER stress sensor, blunts p53 induction and rescues development of Rpl22-deficient αβ T cell progenitors. Rpl22 deficiency appears to exacerbate ER stress by interfering with the ability of ER stress signals to block new protein synthesis. Our finding that Rpl22 deficiency exacerbates ER stress responses and induces p53 in αβ T cell progenitors provides insight into how a ubiquitously expressed RP can perform regulatory functions that are selectively required by some cell lineages but not others.

Mutations in ribosomal proteins cause bone marrow failure syndromes associated with increased cancer risk, but the basis by which they do so remains unclear. We reported previously that the ribosomal protein Rpl22 is a tumor suppressor in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), and that loss of just one Rpl22 allele accelerates T-cell lymphomagenesis by activating NF-κB and inducing the stem cell factor Lin28B. Here, we show that, paradoxically, loss of both alleles of Rpl22 restricts lymphoma progression through a distinct effect on migration of malignant cells out of the thymus. Lymphoma-prone AKT2-transgenic or PTEN-deficient mice on an Rpl22−/− background developed significantly larger and markedly more vascularized thymic tumors than those observed in Rpl22+/+ control mice. But, unlike Rpl22+/+ or Rpl22+/− tumors, Rpl22−/− lymphomas did not disseminate to the periphery and were retained in the thymus. We traced the defect in the Rpl22−/− lymphoma migratory capacity to downregulation of the KLF2 transcription factor and its targets, including the key migratory factor sphingosine 1-phosphate receptor 1 (S1PR1). Indeed, reexpression of S1PR1 in Rpl22-deficient tumor cells restores their migratory capacity in vitro. The regulation of KLF2 and S1PR1 by Rpl22 appears to be proximal as Rpl22 reexpression in Rpl22-deficient lymphoma cells restores expression of KLF2 and S1P1R, while Rpl22 knockdown in Rpl22-sufficient lymphomas attenuates their expression. Collectively, these data reveal that, while loss of one copy of Rpl22 promotes lymphomagenesis and disseminated disease, loss of both copies impairs responsiveness to migratory cues and restricts malignant cells to the thymus. Cancer Res; 76(11); 3387–96. ©2016 AACR.

THEMIS, the enigmatic regulator of T cell selection in the thymus, controls selection by oxidizing and suppressing the activity of the tyrosine phosphatase SHP-1.

We have previously demonstrated that loss of the tumor suppressive activity of ribosomal protein (RP) RPL22 predisposes to development of leukemia in mouse models and aggressive disease in human patients; however, the role of RPL22 in solid tumors, specifically colorectal cancer (CRC), had not been explored. We report here that RPL22 is either deleted or mutated in 36% of CRC and provide new insights into its mechanism of action. Indeed, Rpl22 inactivation causes the induction of its highly homologous paralog, RPL22L1, which serves as a driver of cell proliferation and anchorage-independent growth in CRC cells. Moreover, RPL22L1 protein is highly expressed in patient CRC samples and correlates with poor survival. Interestingly, the association of high RPL22L1 expression with poor prognosis appears to be linked to resistance to 5-Fluorouracil, which is a core component of most CRC therapeutic regimens. Indeed, in an avatar trial, we found that human CRC samples that were unresponsive to 5-Fluorouracil in patient-derived xenografts exhibited elevated expression levels of RPL22L1. This link between RPL22L1 induction and 5-Fluorouracil resistance appears to be causal, because ectopic expression or knockdown of RPL22L1 in cell lines increases and decreases 5-Fluorouracil resistance, respectively, and this is associated with changes in expression of the DNA-repair genes, MGMT and MLH1. In summary, our data suggest that RPL22L1 might be a prognostic marker in CRC and predict 5-FU responsiveness.

γδ NKT cells are neonatal-derived γδ T lymphocytes that are grouped together with invariant NKT cells based on their shared innate-like developmental program characterized by the transcription factor PLZF (promyelocytic leukemia zinc finger). Previous studies have demonstrated that the population size of γδ NKT cells is tightly controlled by Id3-mediated inhibition of E-protein activity in mice. However, how E proteins promote γδ NKT cell development and expansion remains to be determined. In this study, we report that the transcription factor Egr2, which also activates PLZF expression in invariant NKT cells, is essential for regulating γδ NKT cell expansion. We observed a higher expression of Egr family genes in γδ NKT cells compared with the conventional γδ T cell population. Loss of function of Id3 caused an expansion of γδ NKT cells, which is accompanied by further upregulation of Egr family genes as well as PLZF. Deletion of Egr2 in Id3-deficient γδ NKT cells prevented cell expansion and blocked PLZF upregulation. We further show that this Egr2-mediated γδ NKT cell expansion is dependent on c-Myc. c-Myc knockdown attenuated the proliferation of Id3-deficient γδ NKT cells, whereas c-Myc overexpression enhanced the proliferation of Id3/Egr2–double-deficient γδ NKT cells. Therefore, our data reveal a regulatory circuit involving Egr2–Id3–E2A, which normally restricts the population size of γδ NKT cells by adjusting Egr2 dosage and c-Myc expression.

In our recent publication (1), we report that the EGYEL motif, previously found to promote T22-reactivity, was depleted from the mature CD24low pool of γδ-T cells that developed in H-2T–deficient mice, indicating that ligand was influencing the γδ-T cell receptor (TCR) repertoire (1). In her letter, Chien (2) suggests that this conclusion was invalid because, in reporting our analysis, we did not include the upstream tryptophan (W) residue in the motif, which she contends is critical for T22-reactivity (2). We have reanalyzed the data, which reveal that the W…EGYEL motif and all permutations thereof, are depleted from the CD24low population that developed in H-2T–deficient mice (Fig. 1). This motif is found at background levels in tetramer− thymic γδ cells, … [↵][1]1To whom correspondence should be addressed. Email: David.Wiest{at}fccc.edu. [1]: #xref-corresp-1-1

Although Appl1 and Appl2 have been implicated in multiple cellular activities, we and others have found that Appl1 is dispensable for mouse embryonic development, suggesting that Appl2 can substitute for Appl1 during development. To address this possibility, we generated conditionally targeted Appl2 mice. We found that ubiquitous Appl2 knockout (Appl2-/-) mice, much like Appl1-/- mice, are viable and grow normally to adulthood. Intriguingly, when Appl1-/- mice were crossed with Appl2-/- mice, we found that homozygous Appl1;Appl2 double knockout (DKO) animals are also viable and grossly normal with regard to reproductive potential and postnatal growth. Appl2-null and DKO mice were found to exhibit altered red blood cell physiology, with erythrocytes from these mice generally being larger and having a more irregular shape than erythrocytes from wild type mice. Although Appl1/2 proteins have been previously shown to have a very strong interaction with phosphatidylinositol-3 kinase (Pi3k) in thymic T cells, Pi3k-Akt signaling and cellular differentiation was unaltered in thymocytes from Appl1;Appl2 (DKO) mice. However, Appl1/2-null mouse embryonic fibroblasts exhibited defects in HGF-induced Akt activation, migration, and invasion. Taken together, these data suggest that Appl1 and Appl2 are required for robust HGF cell signaling but are dispensable for embryonic development and reproduction.

Although ribosomal proteins facilitate the ribosome’s core function of translation, emerging evidence suggests that some ribosomal proteins are also capable of performing tissue-restricted functions either from within specialized ribosomes or from outside of the ribosome. In particular, we have previously demonstrated that germline ablation of the gene encoding ribosomal protein Rpl22 causes a selective and p53-dependent arrest of αβ T cell progenitors at the β-selection checkpoint. We have now identified a crucial role for Rpl22 during early B cell development. Germline ablation of Rpl22 results in a reduction in the absolute number of B-lineage progenitors in the bone marrow beginning at the pro–B cell stage. Although Rpl22-deficient pro–B cells are hyporesponsive to IL-7, a key cytokine required for early B cell development, the arrest of B cell development does not result from disrupted IL-7 signaling. Instead, p53 induction appears to be responsible for the developmental defects, as Rpl22 deficiency causes increased expression of p53 and activation of downstream p53 target genes, and p53 deficiency rescues the defect in B cell development in Rpl22-deficient mice. Interestingly, the requirement for Rpl22 in the B cell lineage appears to be developmentally restricted, because Rpl22-deficient splenic B cells proliferate normally in response to Ag receptor and Toll receptor stimuli and undergo normal class-switch recombination. These results indicate that Rpl22 performs a critical, developmentally restricted role in supporting early B cell development by preventing p53 induction.

IL-17-producing γδ T (γδT17) cells are critical components of the innate immune system. However, the gene networks that control their development are unclear. Here we show that HEB (HeLa E-box binding protein, encoded by Tcf12) is required for the generation of a newly defined subset of fetal-derived CD73− γδT17 cells. HEB is required in immature CD24+CD73− γδ T cells for the expression of Sox4, Sox13, and Rorc, and these genes are repressed by acute expression of the HEB antagonist Id3. HEB-deficiency also affects mature CD73+ γδ T cells, which are defective in RORγt expression and IL-17 production. Additionally, the fetal TCRγ chain repertoire is altered, and peripheral Vγ4 γδ T cells are mostly restricted to the IFNγ-producing phenotype in HEB-deficient mice. Therefore, our work identifies HEB-dependent pathways for the development of CD73+ and CD73− γδT17 cells, and provides mechanistic evidence for control of the γδT17 gene network by HEB., The γδ T cell pool includes abundant IL-17-producing cells that protect mucosal surfaces, but the signals that control γδ T cell specification are unclear. Here the authors identify a role for the transcription factor HEB, and antagonistic activity of Id3, in the development of these cells.

Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott–Aldrich syndrome protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multisubunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott–Aldrich syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole-exome sequencing revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208VfsX20), in Arp2/3 complex component ARPC1B that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208VfsX20 variant identified in the patients. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, whereas mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed.