ALBERT

Description: Background: Multiple myeloma (MM) is a clonal B cell neoplasia that comes from growth of malignant B cells in the bone marrow. Stromal cells, including inflammatory cells, in the bone marrow enable MM persistence and growth. MM is characterized by an uncoordinated cytokine system with an increase in proinflammatory cytokines. While proinflammatory cytokines are essential in mounting an anti-tumor response, they can also drive cancer progression. Ultimately, it is the effects of the cytokine milieu in the immune microenvironment that help determine MM development. While the role of IFNγ in MM remains mixed and unclear, our analysis suggests IFNγ plays an oncogenic role in MM and offers other insights to MM pathology. Methods: The National Center for Biotechnology Information (NCBI) GEO is an open database of more than 2 million samples of functional genomics experiments. The Search Tag Analyze Resource for GEO (STARGEO) platform allows for meta-analysis of genomic signatures of disease and tissue. We employed the STARGEO platform to search the Gene Expression Omnibus and performed meta-analysis on 517 peripheral blood samples from multiple myeloma patients using 97 healthy peripheral blood samples as a control. We then analyzed the signature in Ingenuity Pathway Analysis (IPA) to help define the genomic signature of MM and identify disease pathways. We analyzed genes that showed statistical significance disease and control samples (p

Description: Long-term murine bone marrow cultures were used to support the growth and development of hematopoietic cells. After hematopoiesis was established, the cultures were infected with a recombinant murine amphotropic virus carrying the avian sarcoma virus src gene and the CFU- S kinetics were examined. The CFU-S from the src-infected cultures displayed a reduced seeding efficiency in the standard spleen colony assay. The self-renewal capacity of these CFU-S was tested by their ability to reestablish hematopoiesis when serially transplanted on irradiated bone marrow cultures and by serial passage in spleens of irradiated mice. In both tests, cells from the src-infected cultures exhibited an enhanced ability to sustain a high level of self-renewal. The other property of stem cells which may be measured is the probability of self-renewal at each cell division which dictates the distribution between stem cells and differentiated type progeny. CFU-S from the src-infected cultures had higher average probabilities of self- renewal and therefore reduced differentiation. These differences suggest that expression of src had indirectly or directly altered the normal differentiation program of the stem cells.

Description: Two potent inhibitors of mono-adenosine diphosphate (ADP) ribosylation have recently been described and characterized, named p- methoxylbenzylaminodecamethylene guanidine sulfate (MBAMG) and benzylaminododecylguanine hydrochloride (BADGH). We have used these agents to investigate the role of ADP ribosylation in hematopoiesis using long-term marrow cultures. The addition of MBAMG (10(-6) mol/L) or BADGH (5 X 10(-4) mol/L) led to both an inhibition of mature cell production and the development of colony-stimulating factor (CSF-1)- responsive GM-CFC, but had no effect upon spleen colony-forming units (CFU-S) or on progenitor cells which respond to the multilineage stimulating factor present in WEHI-3B cell-conditioned medium. These data indicate that these inhibitors of mono-ADP ribosylation can block the commitment and/or differentiation of stem cells and infers that ADP ribosylation may be of some importance in the hematopoietic process.

Description: A recombinant retrovirus (DHFR*-SVADA) in which human adenosine deaminase (ADA) cDNA is transcribed from an internal SV40 promoter was used to infect murine hematopoietic stem and progenitor cells. Human ADA enzyme was not expressed in infected primary murine pluripotent stem cell-derived spleen or progenitor colonies (CFU-GM, CFU-Mix, BFU- E). In contrast, human ADA enzyme activity was readily detected in progenitor colonies derived from immortalized multipotent factor- dependent cells. The level of human enzyme was near endogenous murine enzyme levels and was equivalent in undifferentiated stem cells and differentiated myeloid, erythroid, and mixed colonies. These results indicate that cellular properties other than the stage of differentiation are important in determining the expression of foreign sequences introduced by retroviruses. Cell lines that are immortalized but still capable of induced differentiation may contain factors that abrogate blocks to expression that are manifested in primary hematopoietic stem cells.

Description: A recombinant retrovirus (DHFR*-SVADA) in which human adenosine deaminase (ADA) cDNA is transcribed from an internal SV40 promoter was used to infect murine hematopoietic stem and progenitor cells. Human ADA enzyme was not expressed in infected primary murine pluripotent stem cell-derived spleen or progenitor colonies (CFU-GM, CFU-Mix, BFU- E). In contrast, human ADA enzyme activity was readily detected in progenitor colonies derived from immortalized multipotent factor- dependent cells. The level of human enzyme was near endogenous murine enzyme levels and was equivalent in undifferentiated stem cells and differentiated myeloid, erythroid, and mixed colonies. These results indicate that cellular properties other than the stage of differentiation are important in determining the expression of foreign sequences introduced by retroviruses. Cell lines that are immortalized but still capable of induced differentiation may contain factors that abrogate blocks to expression that are manifested in primary hematopoietic stem cells.

Description: Long-term murine bone marrow cultures were used to support the growth and development of hematopoietic cells. After hematopoiesis was established, the cultures were infected with a recombinant murine amphotropic virus carrying the avian sarcoma virus src gene and the CFU- S kinetics were examined. The CFU-S from the src-infected cultures displayed a reduced seeding efficiency in the standard spleen colony assay. The self-renewal capacity of these CFU-S was tested by their ability to reestablish hematopoiesis when serially transplanted on irradiated bone marrow cultures and by serial passage in spleens of irradiated mice. In both tests, cells from the src-infected cultures exhibited an enhanced ability to sustain a high level of self-renewal. The other property of stem cells which may be measured is the probability of self-renewal at each cell division which dictates the distribution between stem cells and differentiated type progeny. CFU-S from the src-infected cultures had higher average probabilities of self- renewal and therefore reduced differentiation. These differences suggest that expression of src had indirectly or directly altered the normal differentiation program of the stem cells.

Description: Background: Monoclonal gammopathy of undetermined significance (MGUS) is characterized by plasma cell production of abnormal monoclonal protein, or M protein. While MGUS itself is asymptomatic, it generally carries a 1% per year risk to progression to multiple myeloma (MM). The etiology of MGUS, as well as why it progresses to MM in some cases, remains unclear. Moreover, it is not known why some MGUS patients, such as African Americans, have higher risk to progression to MM. Contrasting MGUS and MM can potentially highlight genes that differentiate benign gammopathies from malignant ones and may be involved in disease progression from MGUS to MM. Methods: We employed our STARGEO platform to tag samples from the NCBI Gene Expression Omnibus and performed two separate meta-analysis to compare MGUS and MM transcriptomes. For the first meta-analysis, we tagged MGUS plasma cells recovered from the bone marrow of 101 patients and tagged plasma cells from 64 healthy subjects as a control. For the second analysis. We tagged CD138+ cells from the bone marrow of 383 MM patients and used the MGUS tagged samples as a control. We then analyzed the signature in Ingenuity Pathway Analysis (IPA). Results: From our first meta-analysis of MGUS, we identified EIF2 signaling, regulation of EIF4 and p70S6K signaling, and JAK/STAT signaling as top canonical pathways. Top upstream regulators included TP53, TGFB1, and the proto-oncogene MYCN and MYC (with predicted activation). The most upregulated genes included pro-oncogenes such as KIT and MLLT3, which is well-studied in acute leukemia but not yet described in MGUS. Another top upregulated gene was NRG3, a myeloma growth factor. Additionally, our analysis highlighted key genes involved in transcription and epigenetic regulation. For example, there was upregulation of RBFOX2, which is involved in alternative splicing during oncogenesis and tumor progression, and of PARP15, a transcriptional repressor with poly(ADP-ribose) polymerase activity and candidate gene for drug targeting. Also, there was upregulation of the DNA damage-inducible gene GADD45A, found to promote global DNA methylation. Lastly, we found upregulation of COMMD3, a gene with a recently identified role in humoral activity and B cell migration. From our second meta-analysis comparing MM and MGUS directly, we identified mitochondrial dysfunction, oxidative phosphorylation, purine nucleotides de novo biosynthesis, and sirtuin signaling as top upstream regulators. Like our first analysis, TP53 (with predicted inhibition), TGFB1, and MYC (with predicted activation) were top upstream regulators. The most upregulated gene was NUP62, a nucleoporin and novel regulator of cell proliferation and inducer of MYC activity. Our analysis also illustrated pro-oncogenic signaling pathways such as the Wnt pathway through upregulation of the ubiquitin ligase RNF14 and serine/threonine kinase through upregulation of SRPK2. Moreover, we found upregulation of the super-enhancer DUSP4, a phosphatase whose over-activity may drive MM severity. Lastly, we found upregulation of lysosomal associated membrane protein LAMP5. LAMP5 was recently identified in single-cell RNA sequencing of MM patients and may play a significant role in disease. Conclusions: Our study illustrates signaling pathways in MGUS that are present in MM such as EIF2, JAK/STAT, and MYC signaling. We also illustrate gene activity in MGUS that may predispose to MM progression such as NRG3, RBFOX2, and PARP15. GADD45RA and COMMD3 may play novel roles in MGUS. Our second analysis highlighted disease activity that persist from MGUS to MM, such as MYC signaling. It is possible that the genes from this analysis that aims to distinguish MM from MGUS may be responsible for tipping the scales from benignity to malignancy. Genes such as DUSP4, RN14, LAMP5, and others could serve as novel biomarkers or targets to MM and risk of progression of MGUS to MM. Disclosures No relevant conflicts of interest to declare.