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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.

BRCA1-Delta11q mRNA and protein isoform.

Ectopic BRCA1-Deltaq expression promotes resistance in additional cell line backgrounds.

CRISPR/Cas9 editing of SUM149PT cells.

FOX2 binding motifs are proximal to the BRCA1-11q cryptic slice site.

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).

BRCA1-minigene analyses.

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

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.

Inactivation of ribosomal proteins (RP) is known to cause diseases called ribosomopathies, which are often associated with abnormal hematopoiesis and an increased risk for development of leukemia. Our laboratory has recently reported an important link between inactivation of one such ribosomal protein, Rpl22, and poor survival in T cell acute lymphoblastic leukemia (T-ALL) patients. Preliminary bioinformatic analysis shows that RPL22 is lost in ∼10% of pediatric T-ALL patients, who exhibit a more aggressive disease course. Deletion of the RPL22 locus is also enriched in the early T-cell precursor (ETP-ALL) subset of T-ALL patients, which exhibit a more aggressive disease course. Thus, RPL22 loss appears to be a marker for poor outcome in pediatric T-ALL patients. While RP inactivation has previously been linked to increased cancer risk, the mechanistic basis for this linkage remained unclear. We have recently demonstrated that inactivation of Rpl22 promotes leukemic transformation by activating NF-κB and inducing the stem cell gene, LIN28B. Nevertheless, the molecular link between Rpl22 inactivation and the induction of NF-κB activity remained unclear. We have now determined that inactivation of Rpl22 activates NF-κB signaling by exacerbating ER stress responses. Indeed, Rpl22 loss results in hyperactivation of the PERK-EIF2α-ATF4 stress pathway, which is known to activate NF-κB. We have demonstrated that knockdown of PERK using shRNA abrogates the activation of NF-κB in Rpl22 mutant cells and returns Lin28B expression levels to baseline. This reduction in Lin28B eliminates the predisposition to transformation exhibited by Rpl22 mutant cells, as evidenced by decreased formation of colonies in soft agar. Taken together, these data suggest that RPL22 inactivation may serve as a negative prognostic indicator in T-ALL and that pharmacologic targeting of ER stress pathways may represent a novel therapeutic alternative in this molecularly-defined subclass of T-ALL. Citation Format: Nehal R. Solanki-Patel, Noa Greenberg, Shu Rao, Suraj Peri, Michael Slifker, Charles Mullighan, David Wiest. Role of ribosomal protein, Rpl22 in regulation of acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2864.

Inactivation of ribosomal proteins (RP) is known to cause diseases called ribosomopathies, which are often associated with abnormal hematopoiesis and an increased risk for development of leukemia. Our laboratory has recently reported an important link between inactivation of one such ribosomal protein, Rpl22, and poor survival in T cell acute lymphoblastic leukemia (T-ALL) patients. Preliminary bioinformatic analysis shows that RPL22 is lost in ~10% of pediatric T-ALL patients, who exhibit a more aggressive disease course. Deletion of the RPL22 locus is also enriched in the early T-cell precursor (ETP-ALL) subset of T-ALL patients, which exhibit a more aggressive disease course. Thus, RPL22 loss appears to be a marker for poor outcome in pediatric T-ALL patients. While RP inactivation has previously been linked to increased cancer risk, the mechanistic basis for this linkage remained unclear. We have recently demonstrated that inactivation of Rpl22 promotes leukemic transformation by activating NF-kB and inducing the stem cell gene, LIN28B. Nevertheless, the molecular link between Rpl22 inactivation and the induction of NF-kB activity remained unclear. We have now determined that inactivation of Rpl22 activates NF-kB signaling by exacerbating ER stress responses. Indeed, Rpl22 loss results in hyperactivation of the PERK-EIF2α-ATF4 stress pathway, which is known to activate NF-kB. We have demonstrated that knockdown of PERK using shRNA abrogates the activation of NF-kB in Rpl22 mutant cells and returns Lin28B expression levels to baseline. This reduction in Lin28B eliminates the predisposition to transformation exhibited by Rpl22 mutant cells, as evidenced by decreased formation of colonies in soft agar. Taken together, these data suggest that RPL22 inactivation may serve as a negative prognostic indicator in T-ALL and that pharmacologic targeting of ER stress pathways may represent a novel therapeutic alternative in this molecularly-defined subclass of T-ALL. Citation Format: Nehal Solanki Patel, Noa Greenberg, Suraj Peri, Shuyun Rao, Michele Rhodes, David Wiest. Role of ribosomal protein, Rpl22 in regulating leukemic transformation. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B26.

Inactivation of ribosomal proteins (RP) is known to cause diseases called ribosomopathies, which are often associated with abnormal hematopoiesis and an increased risk for development of leukemia. Our laboratory has recently reported an important link between inactivation of one such ribosomal protein, Rpl22, and poor survival in T cell acute lymphoblastic leukemia (T-ALL) patients. Preliminary bioinformatic analysis shows that RPL22 is lost in ∼10% of pediatric T-ALL patients, who exhibit a more aggressive disease course. Deletion of the RPL22 locus is also enriched in the early T-cell precursor (ETP-ALL) subset of T-ALL patients, which exhibit a more aggressive disease course. Thus, RPL22 loss appears to be a marker for poor outcome in pediatric T-ALL patients. While RP inactivation has previously been linked to increased cancer risk, the mechanistic basis for this linkage remained unclear. We have recently demonstrated that inactivation of Rpl22 promotes leukemic transformation by activating NF-κB and inducing the stem cell gene, LIN28B. Nevertheless, the molecular link between Rpl22 inactivation and the induction of NF-κB activity remained unclear. We have now determined that inactivation of Rpl22 activates NF-κB signaling by exacerbating ER stress responses. Indeed, Rpl22 loss results in hyperactivation of the PERK-EIF2a-ATF4 stress pathway, which is known to activate NF-κB. We have demonstrated that knockdown of PERK using shRNA abrogates the activation of NF-κB in Rpl22 mutant cells and returns Lin28B expression levels to baseline. This reduction in Lin28B eliminates the predisposition to transformation exhibited by Rpl22 mutant cells, as evidenced by decreased formation of colonies in soft agar. Taken together, these data suggest that RPL22 inactivation may serve as a negative prognostic indicator in T-ALL and that pharmacologic targeting of ER stress pathways may represent a novel therapeutic alternative in this molecularly-defined subclass of T-ALL. Citation Format: Nehal Solanki Patel, Noa Greenberg Kushnir, Shuyun Rao, Suraj Peri, Michele Rhodes, Panduka Nagahawatte, Charles Mullighan, David L. Wiest. Role of ribosomal protein, Rpl22 in regulating leukemic transformation. [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 1269. doi:10.1158/1538-7445.AM2015-1269

Mutations in ribosomal proteins (RP) often cause bone marrow syndromes associated with increased risk for cancer development. We have shown that Rpl22 is a haploinsufficient tumor suppressor in T acute lymphoblastic leukemia (T-ALL). Indeed, loss of one Rpl22 allele accelerates T-cell lymphomagenesis in the murine Akt transgenic mouse model, by inducing the stem cell factor Lin28B in an NFκB-dependent manner. Moreover, mice lacking both Rpl22 alleles exhibited significantly increased thymic tumor size, which was associated with poor survival and markedly enhanced angiogenesis relative to Rpl22 heterozygous mice or wild type. The increased angiogenesis in the mediastinal mass was linked to upregulation of VEGFA and downregulation of S1P1. Loss of S1P1 is sufficient to block egress of lymphoma cells from thymus. Rpl22-deficient thymic lymphoma lines also exhibit enhanced growth upon adaptation to growth in vitro, and this is reversed by ectopic expression of Rpl22. Rpl22 expression also represses the expression of proteins that promote traversal of the cell cycle. Collectively, the increased tumor growth and angiogenesis, and the blockade of thymic egress contribute to the enlarged mediastinal masses observed in Rpl22-deficient mice. Rpl22-mutant T-ALL are a molecularly distinct subtype exhibiting increased aggression and treatment resistance. Accordingly, our identification of a number of signaling pathways controlled by Rpl22 may enable the development of novel strategies, such as targeting angiogenesis, in such cases of T-ALL. Citation Format: Shuyun Rao, Jason E. Stadanlick, Kathy Q. Cai, David L. Wiest. Loss of Rpl22 promotes tumor progression through regulation of angiogenesis. [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 5215. doi:10.1158/1538-7445.AM2015-5215

The molecular basis by which mutations in some ribosomal proteins (RP) predispose to development of hematologic malignancies remains unclear. We show here that germline inactivation of the ribosomal protein L22 (Rpl22), which selectively blocks T cell development, also predisposes T lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ∼9% of human T-acute lymphoblastic leukemias (T-ALL). Moreover, mono-allelic loss of Rpl22 accelerates development of thymic lymphoma in a mouse model of T cell malignancy and in acute transformation assays in vitro. Furthermore, we show that Rpl22 inactivation enhances transformation potential through induction of Lin28B. These findings represent the first mechanistic insight into how an RP mutation promotes transformation.Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides insight into the mechanistic basis by which mutations in Rpl22, and perhaps other RP genes, increase cancer risk. In addition, because we have determined that Lin28B induction is dependent upon NF-kB activity, our findings may provide new opportunities for therapeutic intervention both in aggressive forms of T-ALL, where we have found Rpl22 to be inactivated, and perhaps in other diseases caused by mutations in RP genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1353. doi:1538-7445.AM2012-1353