Cancer gene discovery has relied extensively on analyzing tumors for gains and losses to reveal the location of oncogenes and tumor suppressor genes, respectively. Deletions of 1p36 are extremely common genetic lesions in human cancer, occurring in malignancies of epithelial, neural, and hematopoietic origin. Although this suggests that 1p36 harbors a gene that drives tumorigenesis when inactivated, the identity of this tumor suppressor has remained elusive. Here we use chromosome engineering to generate mouse models with gain and loss of a region corresponding to human 1p36. This approach functionally identifies chromodomain helicase DNA binding domain 5 (Chd5) as a tumor suppressor that controls proliferation, apoptosis, and senescence via the p19(Arf)/p53 pathway. We demonstrate that Chd5 functions as a tumor suppressor in vivo and implicate deletion of CHD5 in human cancer. Identification of this tumor suppressor provides new avenues for exploring innovative clinical interventions for cancer.

Interactions of Artemis with DNA Ligase IV and DNA-PKcs are required for efficient coding joint formation.

The pathway of V(D)J recombination was discovered almost three decades ago. Yet it continues to baffle scientists because of its inherent complexity and the multiple layers of regulation that are required to efficiently generate a diverse repertoire of T and B cells. The non-homologous end-joining (NHEJ) DNA repair pathway is an integral part of the V(D)J reaction, and its numerous players perform critical functions in generating this vast diversity, while ensuring genomic stability. In this review, we summarize the efforts of a number of laboratories including ours in providing the mechanisms of V(D)J regulation with a focus on the NHEJ pathway. This involves discovering new players, unraveling unknown roles for known components, and understanding how deregulation of these pathways contributes to generation of primary immunodeficiencies. A long-standing interest of our laboratory has been to elucidate various mechanisms that control RAG activity. Our recent work has focused on understanding the multiple protein-protein interactions and protein-DNA interactions during V(D)J recombination, which allow efficient and regulated generation of the antigen receptors. Exploring how deregulation of this process contributes to immunodeficiencies also continues to be an important area of research for our group.

DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620 to 800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4.

Despite important advances in therapeutic options accounting for improved outcomes in childhood cancers, there remains a significant subset of patients with hematologic malignancies for whom outcomes remain dismal. Notably among this group are patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). DLBCL accounts for approximately 10-20% of pediatric Non-Hodgkin’s Lymphoma (NHL) cases and with intensive multiagent chemotherapy the 5-year event free survival is ~90%. However, for those with progressive or relapsed disease, survival is dismal at <30% and remains a clinical challenge even with use of intensive salvage regimens. As such, identifying novel agents that will increase survival in this group highlights an unmet need. Siglec-15, a member of the sialic acid binding immunoglobulin-like lectin family of proteins, has recently been identified as a critical immune suppressor that is highly expressed in human cancers and intra-tumoral myeloid cells. Importantly, inhibiting Siglec-15, either through genetic knockout or knockdown, had a restorative effect on local anti-tumor immune responses and abrogated tumor progression. While reported in solid malignancies, a role for Siglec-15 in promoting disease progression in hematologic malignancies has not yet been described. We have evaluated Siglec-15 expression in primary human lymphoma patient samples as well as various lymphoma (human and mouse) cell lines using western blot, quantitative PCR as well immunohistochemistry and immunofluorescence methods. We are evaluating the effect of inhibiting expression of Siglec-15 through genetic downregulation in human lymphoma cell lines as well as the well-established murine lymphoma cell line A20 on T cell function and proliferation using methods such as flow cytometry and ELISA. Analyses of public RNA-seq data sets demonstrates higher Sig15 expression in DLBCL cells compared to normal B cells. Western blot shows higher Sig15 expression in lymphoma cell lines compared to PBMC. IHC of a tumor microarray and validation samples from children shows high Sig15 expression in NHL samples with distinct staining patterns based on subtype. Specifically, Sig15 appears to be highly expressed and associated with the cell membrane in most DLBCL and Burkitt’s lymphoma, with more variable expression in anaplastic large cell lymphoma, primarily in the cytoplasm at low levels and/or in cells with morphology consistent with macrophages. In addition, preliminary data suggest more than one isoform of Sig15, raising the possibility of alternative functions. Lastly, knockdown of Sig15 in A20 cells abrogates disease progression in immune competent but not immune deficient recipients, consistent with a role for Sig15 in immune evasion in lymphoma. Together, these data implicate Sig15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to lymphoma cells. Citation Format: Dailia B. Francis, Jodi Dougan, Claire Pillsbury, Sunita Park, Linda Liu, Christopher Porter. Elucidating the role and mechanisms by which siglec-15 promotes immune dysregulation in lymphoma [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 3845.