Oncolytic HSV Vectors and Anti-Tumor Immunity

The therapeutic promise of oncolytic viruses (OVs) rests on their ability to both selectively kill tumor cells and induce anti-tumor immunity. The potential of tumors to be recognized and eliminated by an effective anti-tumor immune response has been spurred on by the discovery that immune checkpoint inhibition can overcome tumor-specific cytotoxic T cell (CTL) exhaustion and provide durable responses in multiple tumor indications. OV-mediated tumor destruction is now recognized as a powerful means to assist in the development of anti-tumor immunity for two important reasons: (i) OVs, through the elicitation of an anti-viral response and the production of type I interferon, are potent stimulators of inflammation and can be armed with transgenes to further enhance anti-tumor immune responses; and (ii) lytic activity can promote the release of tumor-associated antigens (TAAs) and tumor neoantigens that function as in situ tumor-specific vaccines to elicit adaptive immunity. Oncolytic herpes simplex viruses (oHSVs) are among the most widely studied OVs for the treatment of solid malignancies, and Amgen's oHSV Imlygic® for the treatment of melanoma is the only OV approved in major markets. Here we describe important biological features of HSV that make it an attractive OV, clinical experience with HSV-based vectors, and strategies to increase applicability to cancer treatment

Sequential and Coordinated Actions of c-Myc and N-Myc Control Appendicular Skeletal Development

BACKGROUND: During limb development, chondrocytes and osteoblasts emerge from condensations of limb bud mesenchyme. These cells then proliferate and differentiate in separate but adjacent compartments and function cooperatively to promote bone growth through the process of endochondral ossification. While many aspects of limb skeletal formation are understood, little is known about the mechanisms that link the development of undifferentiated limb bud mesenchyme with formation of the precartilaginous condensation and subsequent proliferative expansion of chondrocyte and osteoblast lineages. The aim of this study was to gain insight into these processes by examining the roles of c-Myc and N-Myc in morphogenesis of the limb skeleton. METHODOLOGY/PRINCIPAL FINDINGS: To investigate c-Myc function in skeletal development, we characterized mice in which floxed c-Myc alleles were deleted in undifferentiated limb bud mesenchyme with Prx1-Cre, in chondro-osteoprogenitors with Sox9-Cre and in osteoblasts with Osx1-Cre. We show that c-Myc promotes the proliferative expansion of both chondrocytes and osteoblasts and as a consequence controls the process of endochondral growth and ossification and determines bone size. The control of proliferation by c-Myc was related to its effects on global gene transcription, as phosphorylation of the C-Terminal Domain (pCTD) of RNA Polymerase II, a marker of general transcription initiation, was tightly coupled to cell proliferation of growth plate chondrocytes where c-Myc is expressed and severely downregulated in the absence of c-Myc. Finally, we show that combined deletion of N-Myc and c-Myc in early limb bud mesenchyme gives rise to a severely hypoplastic limb skeleton that exhibits features characteristic of individual c-Myc and N-Myc mutants. CONCLUSIONS/SIGNIFICANCE: Our results show that N-Myc and c-Myc act sequentially during limb development to coordinate the expansion of key progenitor populations responsible for forming the limb skeleton

Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites.

The small constitutively expressed bHLHZip protein Max is known to form sequence-specific DNA binding heterodimers with members of both the Myc and Mad families of bHLHZip proteins. Myc:Max complexes activate transcription, promote proliferation, and block terminal differentiation. In contrast, Mad:Max heterodimers act as transcriptional repressors, have an antiproliferative effect, and are induced upon differentiation in a wide variety of cell types. We have identified a novel bHLHZip Max-binding protein, Mnt, which belongs to neither the Myc nor the Mad families and which is coexpressed with Myc in a number of proliferating cell types. Mnt:Max heterodimers act as transcriptional repressors and efficiently suppress Myc-dependent activation from a promoter containing proximal CACGTG sites. Transcription repression by Mnt maps to a 13-amino-acid amino-terminal region related to the Sin3 interaction domain (SID) of Mad proteins. We show that this region of Mnt mediates interaction with mSin3 corepressor proteins and that its deletion converts Mnt from a repressor to an activator. Furthermore, wild-type Mnt suppresses Myc+Ras cotransformation of primary cells, whereas Mnt containing a SID deletion cooperates with Ras in the absence of Myc to transform cells. This suggests that Mnt and Myc regulate an overlapping set of target genes in vivo. When mnt is expressed as a transgene under control of the beta-actin promoter in mice the transgenic embryos exhibit a delay in development and die during mid-gestation, when c- and N-Myc functions are critical. We propose that Mnt:Max:Sin3 complexes normally function to restrict Myc:Max activities associated with cell proliferation

Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis.

The Mad family comprises four basic-helix-loop-helix/leucine zipper proteins, Mad1, Mxi1, Mad3, and Mad4, which heterodimerize with Max and function as transcriptional repressors. The balance between Myc-Max and Mad-Max complexes has been postulated to influence cell proliferation and differentiation. The expression patterns of Mad family genes are complex, but in general, the induction of most family members is linked to cell cycle exit and differentiation. The expression pattern of mad3 is unusual in that mad3 mRNA and protein were found to be restricted to proliferating cells prior to differentiation. We show here that during murine development mad3 is specifically expressed in the S phase of the cell cycle in neuronal progenitor cells that are committed to differentiation. To investigate mad3 function, we disrupted the mad3 gene by homologous recombination in mice. No defect in cell cycle exit and differentiation could be detected in mad3 homozygous mutant mice. However, upon gamma irradiation, increased cell death of thymocytes and neural progenitor cells was observed, implicating mad3 in the regulation of the cellular response to DNA damage

Sequential expression of the MAD family of transcriptional repressors during differentiation and development.

Members of the Myc proto-oncogene family encode transcription factors that function in multiple aspects of cell behavior, including proliferation, differentiation, transformation and apoptosis. Recent studies have shown that MYC activities are modulated by a network of nuclear bHLH-Zip proteins. The MAX protein is at the center of this network in that it associates with MYC as well as with the family of MAD proteins: MAD1, MXI1, MAD3 and MAD4. Whereas MYC-MAX complexes activate transcription, MAD-MAX complexes repress transcription through identical E-box binding sites. MAD proteins therefore act as antagonists of MYC. Here we report the expression patterns of the Mad gene family in the adult and developing mouse. High level of Mad gene expression in the adult is limited to tissues that display constant renewal of differentiated cell populations. In embryos, Mad transcripts are widely distributed with expression peaking during organogenesis at the onset of differentiation. A detailed analysis of their pattern of expression during chrondrocyte and neuronal differentiation in vivo, and during neuronal differentiation of P19 cells in vitro, shows that Mad family genes are sequentially induced. Mad3 transcripts and proteins are detected in proliferating cells prior to differentiation. Mxi1 and Mad4 transcripts are most abundant in cells that have further advanced along the differentiation pathway, whereas Mad1 is primarily expressed late in differentiation. Taken together, our data suggest that the different members of the MAD protein family exert their functions at distinct steps during the transition between proliferation and differentiation

Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation.

The switch from transcriptionally activating MYC-MAX to transcriptionally repressing MAD1-MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1-MAX dimers during differentiation, we disrupted the Mad1 gene by homologous recombination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals revealed that cell cycle exit of granulocytic precursors was inhibited following the colony-forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster-forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes were essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes was correlated with a compensatory decrease in granulocytic cluster-forming cell survival under apoptosis-inducing conditions. In addition, recovery of the peripheral granulocyte compartment following bone marrow ablation was significantly enhanced in Mad1 knockout mice. Two Mad1-related genes, Mxi1 and Mad3, were found to be expressed ectopically in adult spleen, indicating that functional redundancy and cross-regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation

Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation.

The basic helix-loop-helix-leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1. Whereas both Myc:Max and Mad:Max heterodimers bind related E-box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation. Here we report the identification and characterization of two novel Mad1- and Mxi1-related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c-Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth-arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co-expressed with either c-myc, N-myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues