NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-β. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.

The t(8;21)(q22;q22) translocation, present in ~5% of adult acute myeloid leukemia (AML) cases, produces the AML1/ETO fusion protein. Dysregulation of the POU domain-containing transcription factor POU4F1 is a recurring abnormality in t(8;21) AML. Here, we show that POU4F1 over-expression is highly correlated with, but not caused by AML1/ETO. AML1/ETO markedly increases the self-renewal capacity of myeloid progenitors from murine bone marrow or fetal liver and drives expansion of these cells in liquid culture. POU4F1 is neither necessary nor sufficient for these AML1/ETO-dependent properties, suggesting that it contributes to leukemia through novel mechanisms. To identify targets of POU4F1, we performed gene expression profiling in primary mouse cells with genetically defined levels of POU4F1 and identified 140 differentially expressed genes. This expression signature was significantly enriched in human t(8;21) AML samples and was sufficient to cluster t(8;21) AML samples in an unsupervised hierarchical analysis. Among the most highly differentially expressed genes, half are known AML1/ETO targets, implying that the unique transcriptional signature of t(8;21) AML is, in part, attributable to POU4F1 and not AML1/ETO itself. These genes provide novel candidates for understanding the biology and developing therapeutic approaches for t(8;21) AML.

Primary human AML cells can be isolated and studied in vitro, but many experimental questions can only be addressed using in vivo models. In particular, tractable animal models are needed to test novel therapies. The genetic complexity of human AML poses significant challenges for the generation of reliable animal models. The hematopoietic systems of both zebrafish ( Danio rerio) and Drosophila have been well characterized ( reviewed in [5, 31]) . Both organisms are well suited to forward genetics mutagenesis screens. Although this approach has been useful for identification of mutants with hematopoietic phenotypes ( e.g., cloche), the impact on cancer biology and hematopoietic malignancies in particular has been limited. A zebrafish model of acute lymphoblastic leukemia has been generated [37] and Drosophila models have shed light on the biology of epithelial tumors ( reviewed in [60]). Nonetheless, in vivo modeling of human AML relies most heavily on mice. Most cellular, molecular, and developmental features of the hematopoietic system are well conserved across mammalian species. The availability of the human and mouse genome sequences and the capability of manipulating the mouse genome make mice the most valuable model organism for AML research. Mice have additional practical value because they have a short reproductive cycle and are relatively inexpensive to house.

Multiple myeloma (MM) is a highly refractory hematological cancer. Targeted immunotherapy has shown promise in MM but remains hindered by the challenge of identifying specific yet broadly representative tumor markers. We analyzed 53 bone marrow (BM) aspirates from 41 MM patients using an unbiased, high-throughput pipeline for therapeutic target discovery via single cell transcriptomic profiling, yielding 38 MM marker genes encoding cell-surface proteins and 15 encoding intracellular proteins. Of these, 20 candidate genes were highlighted that are not yet under clinical study, 11 of which are previously uncharacterized as therapeutic targets. The findings were cross-validated using bulk RNA-sequencing, flow cytometry, and proteomic mass spectrometry of MM cell lines and patient BM, demonstrating high overall concordance across data types. Independent discovery using bulk RNA-sequencing reiterated top candidates, further affirming the ability of single cell transcriptomics to accurately capture marker expression despite limitations in sample size or sequencing depth. Target dynamics and heterogeneity were further examined using both transcriptomic and immuno-imaging methods. In summary, this study presents a robust and broadly applicable strategy for identifying tumor markers to better inform the development of targeted cancer therapy.

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