Productive viral infection mimics oncogenic transformation in several respects, and some of the same molecular mechanisms are employed by viruses and cancer cells to disrupt key homeostatic mechanisms. These similarities serve as the foundation for the development of ”oncolytic” viruses that are designed to specifically target and kill cancer cells. Although some targeting strategies involve engineering viruses so that they bind specifically to cancers, an even more attractive approach involves developing viruses that can only replicate in cancer cells that contain specific defects in homeostatic control. For example one of the products of the adenovirus E1B locus is a protein that specifically disrupts p53 function, thereby undermining the host p53-dependent antiviral response that would otherwise result in inhibition of DNA synthesis and/or apoptosis. Mutant forms of adenovirus that lack E1B 55K should only replicate in cells with defective p53 function, i.e., cancers. Several groups have developed E1B mutant adenoviruses for cancer therapy, and promising results have been obtained with several of them, including Onyx Pharmaceuticals’. Another promising approach exploits the presence of mutant active Ras.

Rhabdoviruses are RNA viruses that are also being developed as oncolytic agents. Their tumor selectivity is related largely to the fact that tumor cells are often resistant to the antiviral effects of type I interferons (IFNs), which can completely suppress viral replication in normal cells. Eliminating viral mechanisms that suppress autocrine IFN production enhances oncolytic activity while further reducing toxicity to normal host tissues. The investigators designed a synthetic lethal RNAi screen to identify cytoprotective pathways that limit tumor cell killing induced by the Maraba rhabdovirus in three different human cancer cell lines. Their ”hits” were enriched for genes that function within two of the three major pathways that respond to endoplasmic reticular (ER) stress, commonly referred to as the unfolded protein response (UPR). More specifically, the screen implicated the ATF6 and IRE1/XBP1 pathways, as well as downstream genes involved in the transport of protein aggregates out of the ER to the proteasome, in cytoprotection. Importantly, the group also identified a novel small molecule inhibitor of IRE1 that also sensitized tumor but not normal cells to the oncolytic effects of the virus in vitro and in xenografts.

Therefore, if the inhibitor can be further optimized to increase its potency, there is a good chance that these preclinical observations can be translated in patients with cancer. At first glance it might seem surprising that hits within the PERK/eIF2a arm of the UPR were not identified, but in fact this makes sense. Phosphorylation of eIF2a results in global downregulation of cap-dependent host translation, so viruses have evolved many different mechanisms to prevent eIF2a phosphorylation or its downstream consequences in normal cells. Furthermore, we have observed that many tumor cells fail to display increased eIF2a phosphorylation or translational arrest in response to proteotoxic and ER stress, so this arm of the UPR may be disabled in a large subset of cancers anyway. In these cancers the coupling between the proteasome and autophagy is disrupted, which may also be advantageous for productive viral infection if autophagy plays somerole in limiting it. One might also predict that knockdown of UPR or ER-associated decay (ERAD) components would cause a buildup of protein aggregates within the ER and that subsequent viral infection dramatically exacerbates the situation by overwhelming an already stressed ER-Golgi network with increased protein synthetic load.

Indeed, UPR inhibition did cause features of ER stress in infected cells, but they resolved quickly and did not lead to an obvious increase in the accumulation of protein aggregates, strongly suggesting that the sensitization caused by pretreatment with UPR inhibitors was not caused by this mechanism. Rather, UPR inhibition appeared to ”precondition” the cells to subsequent virus-induced cell death by upregulating expression of the caspase adaptor protein, RAIDD, and promoting activation of caspase-2, and knockdown of caspase-2 almost completely rescued the synthetic lethal interaction between UPR inhibition and viral infection. Recent work from Doug Green’s group demonstrated that RAIDD-mediated caspase-2 activation is controlled by the stress-responsive transcription factor, HSF-1, suggesting that heatshocked proteins and/or other (perhaps ER-based?) molecular chaperones may play central roles in controlling stressinduced caspase-2 activation.

Left unresolved are the molecular mechanisms that link UPR inhibition to RAIDD upregulation and viral infection to caspase-2 activation. It does seem likely that some (possibly subtle) perturbation of protein aggregate clearance plays a role, but how, and especially why, this low-level stress, that appears to be completely resolved prior to viral infection, sets the stage for subsequent apoptosis awaits further investigation.

The myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid malignancies characterized by clonal hematopoiesis, impaired differentiation, peripheral blood cytopenias, and increased risk of progression to acute myeloid leukemia. Although recent studies have identified recurrent somatic mutations in most patients with MDS, approximately 20% of patients with MDS had no known somatic genetic or cytogenetic abnormalities in the largest studies to date. Two recent studies report the results of whole-exome sequencing in patients with MDS. Notably, themost frequent novel recurrent mutations found occurred in genes encoding members of the RNA-splicing machinery. The paradigm that alterations in splicing contribute to the pathogenesis of human disease and promote tumorigenesis is well described. However, the majority of disease-associated splicing abnormalities discovered previously were in cis-acting elements that disrupt splice site selection at specific loci.

By contrast identified mutations in the trans-acting members of the spliceosome necessary for processing pre-mRNA to mature mRNA. The genetic data supporting these mutations as disease alleles are compelling; the majority of the mutations in SF3B1 and all of the mutations in U2AF35 and SRSF2 are recurrent, heterozygous point mutations, suggesting a gain of function conferred by these recurrent mutations. In contrast rarer mutations in ZRSR2 and PRPF40B occurred as missense or nonsense mutations, suggesting that these mutations might result in loss of function. In addition, it was found that spliceosomal gene mutations are largely mutually exclusive of one another, consistent with a general role of spliceo some mutations in MDS pathogenesis.In order to understand the spectrum of spliceosomal gene mutations, both groups also sequenced a spectrum of myeloid malignancies in addition to MDS. These data led both groups to note a striking association between SF3B1 mutations and MDS characterized by the presence of ring sideroblasts (RS). Although rare SF3B1 mutations have been reported previously in epithelial cancers derived from pancreas, breast, and ovary, SF3B1 mutations occur in the majority of patients with MDS with RS and much less commonly in other hematologic malignancies.

Although mutations in the other spliceosomal components were more common in other subtypes of MDS, the mutations appear to be most enriched in myeloid malignancies with some component of dysplasia, including MDS of all subtypes and chronic myelomonocytic leukemia. It was noted that mutations in SF3B1 in MDS are associated with longer overall and leukemia-free patient survival. Given the already-known favorable prognosis of MDS with RS, studies to identify whether the prognostic effect of these mutations is independent of MDS histopathologic findings are needed. Moreover, previous reports noting splicing alterations in hematologic malignancies, such as the report of frequent missplicing of GSK3b in CML, will need reevaluation to determine if these cancer-specific splicing alterations result from somatic mutations in the spliceosome. To understand the biological consequences of spliceosomal mutations in hematopoiesis, the authors overexpressed wild-type and mutant forms of U2AF35 hematopoietic cells from wild-type mice. Competitive transplantation with similarly transduced control cells revealed a competitive disadvantage with U2AF35 mutant overexpression.

Further work to characterize the effects of these mutations on other aspects of hematopoietic stem cell function including self-renewal, differentiation, and leukemogenesis are needed. Moreover, comparison of the biological effects of expression of recurrent point mutations with downregulation of expression may be very helpful in understanding the biological consequences of spliceosomal component alterations in neoplastic transformation. The mutations in the spliceosomal complex in different myeloid malignancies suggest that these proteins may have distinct functions at different stages of hematopoietic differentiation. Very little is known about the expression of the various Serine/Arginine-rich (SR) proteins in normal and malignant hematopoiesis or about the function of the spliceosome in normal hematopoietic development. Numerous splicing factors have been targeted for constitutional knockout in mice, but these resulted in largely embryonic or perinatal lethality. Conditional gene targeting in a tissue-specific manner has only been carried out for Srsf1 and Srsf2 so far. Mice with cardiac-specific deletion of Srsf1 develop severe dilated cardiomyopathy, leading to death by 6-8 weeks of life, whereas cardiac-specific Srsf2 knockout mice develop a milder cardiomyopathy and have a relatively normal life span.

These results suggest that the SR proteins fulfill specialized, nonredundant functions. Data arguing for a role of spliceosomal components outside of pre-mRNA processing have also come from in vivo modeling. For instance, in vivo analysis of Sf3b1 knockout mice identified genetic intersection with Polycomb Group protein loss, leading to the identification of multiple physical interactions between SF3B1 and members of the PRC1 complex and the BCL6 corepressive complex. Further work to analyze the role of disordered PRC1 activity and BCL6 activity in MDS-RS pathogenesis is now warranted. Studies shows that the effects of expressing U2AF35 in wildtype and mutant forms on gene expression and showed that overexpression of U2AF35 mutants led to a greater frequency of transcripts with unspliced introns and increased expression of members of the nonsense-mediated decay pathway. They concluded that U2AF35 mutations, and possibly other spliceosomal pathway mutations, function in a dominant-negative manner to inhibit normal splicing, a hypothesis requiring further evaluation. Previous studies have noted overexpression of SR family proteins in epithelial cancers, and overexpression of SR proteins (including SRSF1 and SRSF2) leads to cellular transformation ability in other cellular contexts; as such, future studies will need to dissect differences between the role of mutant and wild-type spliceosome proteins in oncogenic transformation.

Identification of splicing factor mutations in MDS may also provide a possibility for therapeutic intervention. An excellent example comes from investigational therapies for the hereditary disorder Duchenne muscular dystrophy (DMD). DMD most commonly results from mutations in a repetitive domain of Dystrophin. Mutations in this domain can be overcome by ”skipping” the mutated exon to generate truncated functional dystrophin protein. Amazingly, a strategy of delivering an antisense oligonucleotide to block an enhancer of exon splicing of the mutated exon and result in a stable mRNA transcript and dystrophin gene product has been utilized successfully in early clinical trials. In addition, compounds that specifically target the SF3A/B subunits of U2 snRNP to result in nuclear export of intron-bearing precursors exist and should be studied further to determine if they interfere with the aberrant splicing due to recurrent mutations in these subunits. These that these two studies have uncovered a novel pathway of importance to myeloid malignancies that may lead to novel therapeutic approaches for patients with MDS.

Neoplastic cells may express membrane-bound molecules, which they did not express before or only in low quantities, or mutated membrane molecules. As a selective response to immune destruction, tumor cells may use several escape strategies, many of which involve down-regulation of Major histocompatibility complex molecules or other molecules implicated in the antigen-presentation pathway. Such tumor cells do not express tumor-specific peptides on the outer membrane, and consequently they cannot be recognized by Cyto toxic lymphocytes(CTL). Tumor cells may also directly inhibit the recognition or the function of immune cells by releasing immune inhibitory molecules.

However, cells other than CTL attack tumor cells: NK cells, polymorphonuclear leukocytes (PMN), and macrophages/DC do not recognize tumor cells via peptides. These cells seem to be involved in the recently described, natural immunity against tumor cells. Moreover, mice deficient in the innate immune system show higher incidence of tumor cell induction and outgrowth compared to wild-type mice. Thus, cells from the innate immune system may play a role both in the destruction of the tumor cells and in the regulation of Major histocompatibility complex expression on cells with which they interact. However, an important question in the discussion is whether the so-called selective response of tumor cells to immune destruction is

(1) a global inductible response of all tumor cells, i.e., the immune system induces the tumor cells to change character in such a way that the cancer cells become less sensitive to the immune effectors, or

(2) a selective response of surviving tumor cells, i.e., the immune effectors kill sensitive tumor cells but not mutated tumorigenic cells or the ones that have changed (down-regulated) certain characters, which render them invulnerable to the immune attack.

To activate the immune system, neoplastic cells must, in addition to the expression of tumor-associated antigens (TAA), induce cellular stress signals, danger signals, or damage-associated signals that alert the innate immune system. Cell death, damage-associated molecular-pattern molecules, and endogenous danger signals are all associated with expression of heat-shock proteins, chromatin-associated protein high-mobility group box 1, and others. This is followed by the expression of “eat me” signals and suppression of “don’t eat me” signals on the “troubled” cells, which are then taken-up by immature Dendritic cells.

The consequence of the dendrocyte “troubled” cells interaction will greatly differ, depending on the necrotic versus apoptotic status of the “troubled” cells. If the “troubled” cells are necrotic, then they release inflammatory molecules that induce the immature dendrocytes to mature and elicit cross-priming of the immune system. Necrotic cell death releases HMGB1 and proteins derived from the tissue injury, such as hyaluronan fragments and nonprotein purinergic molecules such as ATP and uric acid, and induces inflammation due to IL-1b, IFNg, and Tumor necrosis factor. Cell death, damage-associated molecular-pattern molecules activate cells of the innate immune system by triggering TLR or other alarm-signal receptors. In contrast, if the immature Dendritic Cells take up “troubled” cells undergoing apoptotic cell death, they turn into tolerogenic dendrocytes due in part to the activity of caspases that render HMGB1 inactive.

This causes absence of induction of inflammation and no differentiation of immature Dendritic Cells to mature ones. In addition to Cell death, damage-associated molecular-pattern molecules and endogenous danger signals, tumor cells may release effector molecules that stimulate the immune cells to collaborate in tumor growth in the sub-threshold neoplastic states. The complement system, in particular properdin, seems to play an important role in this process by amplifying the production of reactive oxygen and nitrogen species by myeloid-derived suppressor cells. In fact, growing neoplastic cells may be considered by the tissue as a physical wound, and the tissue response to such “intrusion” is wound healing. This means attraction of stromal, endothelial, and epithelial cells, release of chemokines and cytokines as well as molecules of the blood-clotting system. Thus, both normal repair systems, different danger signals, and the immune system may take part in the initiation process from a transformed neoplastic cell to an established, solid tumor.