Tuesday, October 19, 2021

Blood Pressure, Immunity and p53 Checkpoint.


Background

A few chromosome 19 curiosities developed into a deep-dive after looking into the primordial immune complex, the origins of MHC Class I and antigen receptors as revealed by comparative genomics. And the plot thickened because repressors (of endogenous retroviruses) that gained their binding affinity to retrovirus sequences at the same time their targets invaded the human lineage are preferentially located on chromosome 19. Further, the deletion rate in Zinc Finger clusters (ZNF) located around 19p.12 and 19q13.42, particularly between 51,012,739 and 55,620,741 are about twofold higher than the background deletion rate. A lot going on at this very active location which became the background for this article.

At 19q13.42 kallikrein related peptidase (KLK’s), leukocyte immunoglobulin-like receptors (LILR’s) including killer-cell immunoglobulin-like receptor (KIR’s) as well MYADM, an important blood pressure related gene may also provide some clues to immunity variables that originate from or are influenced by this volatile region.

The retrotransposon bombardment of 19q13.42 and double background deletion rate is a significant remnant. However, when evolutionary MHC changed chromosomes ZNF and within its range the chromosome 19 miRNA cluster (C19MC - 53,671,968 and 54,264,387) were still subjected to the deleterious effect of  transposons. Regardless, suppression mechanics have kept epigenetic, regulatory and transcription processes, across gene’s far and wide on the move at a relatively stable rates. For example, reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues, but the effects of suppression may be sufficient to illicit a more permanent natural defense. In any event insertions and DNA damage are closely related and associated with loss of p53 that results in centrosome amplification. 

As cells pass through epithelial to mesenchymal transition (EMT), DNA damage prevents the normal reduction of p53 levels diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53-deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a “differentiation checkpoint” in early endoderm differentiation that alters cell fate in response to DNA damage.


Reproduction, Blood Pressure and NK

In reproduction, some of the 59 known miRNAs from primate-specific C19MC are highly expressed in human placentas and in the serum of pregnant women. They are also packaged into extracellular vesicles of diverse sizes, including exosomes and endow non-trophoblast cells with resistance to a variety of viruses. At least miR-517a-3p (a C19MC from fetal placenta) was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. miRNA's regulate the migration of human trophoblasts and suppress EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype. 

Maternal uterine or decidual Natural Killer cells (dNK) express AT1, AT2, ANP, proteins of Renin Angiotensin System (RAS) suggesting dNK have the potential to contribute to changes in blood pressure that occur between days 5 and 12 of pregnancy in mice. And, pressure related mechanical stretch on endothelial cells interconnects innate and adaptive immune response in hypertension.

Pressure variables in cells and tissues may result from infection, inflammation and membrane stretch, including inner mitochondrial membrane that affects electron transport chain, endoplasmic reticulum, antigen production, presentation and exosome bound p53 / miRNA release.  ANP colocalization to dNK’s suggests that dNK RAS, at day 12 infers a localized RAS related responsiveness. STAT3 in monocytes was activated by increased endothelial stretch and is involved in driving almost all of the pathways that control NK cytolytic activity as well as the reciprocal regulatory interactions between NK cells and other components of the immune system. The crosstalk between STAT3 and p53/RAS signaling controls cancer cell metastasis and cisplatin resistance via the Slug/MAPK/PI3K/AKT-mediated regulation of EMT and autophagy .

The endothelium maintains a barrier between blood and tissue that becomes more permeable during inflammation. MYADM controls endothelial barrier function through ezrin, radixin, and moesin (ERM)-dependent expression of ICAM-1 a paradigmatic adhesion receptor that regulates leukocyte adhesion. The connection between endothelial membrane and cortical actin cytoskeleton appears to modulate the inflammatory response at the blood tissue barrier. 

Educating NK Subsets 

Looking into some of the ~15 genes scattered among C19MC (~sixty miRNA's) between 53,671,968 and 54,264,387;

1. MYADM was one of two blood pressure signature genes differentially expressed for systolic, diastolic blood pressure and hypertension. Of the ~35 identified genes, several more strongly related to immune cell functions including PRF1, GNLY, TAGAP, IL2RB, GZMB and CD97, NKG7, CLC that are located on chromosome 19.

2. PRPF31 is recruited to introns following the attachment of U4 and U6 (spliceosome) RNA’s. Experiments using PRPF31 determined p53 activation is a general consequence of interfering with the spliceosome. 

3. LILRB1 receptor is expressed on immune cells where it binds to MHC class I molecules on antigen-presenting cells and transduces a negative signal that inhibits stimulation of an immune response. LILRB1 has a polymorphic regulatory region that enhances transcription in NK Cells and recruits zinc finger protein YY1. It also educates expanded human NK cells and defines a unique antitumor NK cell subset with potent antibody-dependent cellular cytotoxicity.

Monocyte/macrophage immunoglobulin-like receptors (MIR) genes are closely linked to the KIR gene family and the gene for FcαR at 19q13.4. A mouse sequence related to MIR was discovered and mapped to a region chromosome 7 syntenic with human 19q13.4 where a cluster of genetic loci jointly contributed to the development of both thymic and splenic invariant natural killer T-cell NKT-cell levels. From different mouse strains and across anatomical sites, the cluster included most of the genes located within the C19MC region:– MYADM, CACNG7, VSTM1, TARM1, PRKCC(G), TFPT, NDUFA3, CNOT3, LENG1, TSEN34, RPS9

Four of nineteen knockout genes, that enhanced NK cell function were on chromosome 19 including GSK3 that phosphorylates Mdm2 to regulate p53 abundance, which would contribute to NK enhancement. 

A study of MHC disassortative mating in humans found Israeli’s were more gene similar, but MHC dissimilar than Europeans who were gene dissimilar and MHC dissimilar . Now, a recent study in American Indians found remarkably low KIR and HLA diversity in Amerindians that revealed signatures of strong purifying selection shaping the centromeric KIR region. This narrows to the importance of LILR-KIR region on chromosome19 that codes for the strongest NK cell educator receptors.

p53 regulates exosomes and miRNA’s directly influence NK responsiveness including regulation of dNK during pregnancy. Exosomes regulated by p53 also transfer it and can suppress growth and proliferation of p53 negative cells. Further, miRNA’s, induced by p53 can directly target ULBP2 mRNA and reduce its cell-surface expression.

Disease highlights
 
rs78378222 polymorphism in the 3'-untranslated region of TP53 contributes to development of age-associated cataracts by modifying miRNA-125b-induced apoptosis of lens epithelial cells. miRNA-125b is a novel negative regulator of p53. Deleting PRPF31 activates the p53 pathway and triggers retinal progenitor cells apoptosis. The members of the miR-125 family (miR-125a on chromosome 19q13.4 and miR-125b on chromosome 21q21.1) reside in two distinct human miRNA clusters with the let-7 and miR-99 families and these miRNAs are thus likely co-transcribed.
  
More succinctly, NK cells are alerted to induction of p53 in cancer cells by upregulation of the NKG2D ligands ULBP1 and ULBP2. p53 also induces expression of miR-34a and miR-34c, which target ULBP2 mRNA for destabilization. Observations suggest two possibly contrasting roles for p53 in NKG2DL expression and requires more investigation into how the regulation is fine-tuned. Extending this model to human populations would suggest that p53 must be inactivated among those with a robust NK response (those with B haplotypes). 

Taken together, our data suggest functional interactions between KIR and HLA modify risks of basal cell carcinoma (BCC) and squamous cell carcinoma, and that KIR encoded by the B genes provide selective pressure for altered p53 in BCC tumors. 

Conclusion

The convergence of several important cellular mechanisms that point back to a 19q13.42 address may illustrate ancient and conserved elements that perpetuate and function as integrated biological units effecting blood pressure, reproduction and immunity. Many of these impart education to innate immunity.







Wednesday, July 28, 2021

Life, Dormancy or Death?


Cellular biology is viewed through different lenses, but pregnancy offers a perspective on the invasive origin of cell division, the senescent state and cancer. Pregnancy causes Natural Killer cells of the decidua (dNK) to expand abundantly until they represent as much as 30% of the mucous membranes' cells. NK cells may be induced to expand by invading trophoblasts to realize the dNK trifecta - robust innate immunity that protects the embryo from maternal infection, modulation of trophoblast invasion and driver of vascular remodeling. However, in many cancers expansion of diverse NK populations fails to materialize and missing sub-sets of NK cell diversity provides a path for cancers unchecked growth. 

In decidual cells at the human maternal-fetal interface, CD82 - the metastasis suppressor may participate in intercellular communication with trophoblasts and limit their invasiveness. Trophoblasts enhance adhesiveness of dNK to the decidua's stromal cells, via the CXCL12/CD82/CD29 signaling pathway which contributes to CD56bright NK cell enrichment a necessary element for heathy pregnancy.

CD82 expression is downregulated in tumor progression of many human cancers and strongly correlated with tumor suppressor p53. It can be activated by p53 through a consensus binding sequence in the promoter. In human ovarian cancer a sequential genetic change at the TP53 and the CXCL12 receptors CXCR4  locus occurs during transformation of surface epithelium. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 was10-fold higher compared to that in parental HCT116 cells with functional wild-type p53.

The CXCL12 ligand is unique for its CXCR4 receptor and both are expressed in human first-trimester endometrial epithelial cells (EECs) at the mRNA and protein level. EEC-conditioned medium and recombinant human CXCL12 significantly increased the migration and invasion of EECs. CXCL12 has also been associated with the recruitment of CD56bright CD25+ dNK subsets in early pregnancy's.

CXCR4 is specifically upregulated in the human endometrium during the implantation window and increased immunostaining observed only when a blastocyst is present. CXCR4/CXCL12 not only enhances trophoblast invasiveness, but also limits over-invasiveness by upregulating CD82. CXCR4 activation increases the CXCL12-CXCR4 signaling axis stimulates vascular endothelial growth factor (VEGF) synthesis which induces CXCR4 and CXCL12 production. This synergistic regulation influences placental vascularization. CXCR4 suppresses apoptosis and increases the viability of trophoblasts. 

Undetectable disseminated tumor cells, in different tissue microenvironments restrain or allow the progression of breast cancer in the liver where in dormant milieu's there are selective increases in NK cells. Stroma crosstalk and exit from dormancy follows a marked contraction of the NK cell compartment and concurrent accumulation of activated hepatic stellate cells (aHSCs). Proteomics on liver co-cultures implicate aHSC-secreted CXCL12 in the induction of NK cell quiescence through CXCR4. CXCL12 expression and aHSC abundance are closely correlated in patients with liver metastases and were inversely correlated with NK cell abundance.

The dNK behavior that checks trophoblast invasion and promotes vascularization resembles immediate and invasive new cancers that may occur in cells of any tissue environment. Similarly expansion of resident tissue NK sub-sets in response may be the determiner of life, the shape of next generation cells, dormancy or death.  


Monday, June 28, 2021

Immunity keeping p53 in check!



In a 2012 study on the topology of the human and mouse m6A RNA methylomes Gene Ontology (GO) analysis of differentially expressed genes (DEG's) indicated a noteworthy enrichment of the p53 signaling pathway: 22/23 genes had differentially expressed splice variants, of which 18 were methylated. Moreover, 15 other members of the signaling pathway, which were not significant DEG's, exhibited significant differential isoform expressions. For example, isoforms of MDM4, needed for p53 inactivation were downregulated. Similar pro-apoptotic effects were observed in other pathway genes including MDM2, FAS and BAX. Higher apoptosis rate in HaCaT-T cells resulted with knockdown of m6A subunit METTL3, which also reversed a significant decrease in p53 activity. Modulation of p53 signaling through splicing may be relevant to induction of apoptosis by silencing of METTL3. 

Then, in a 2019 study of arsenite-induced human keratinocyte transformation knockdown of METTL3 significantly decreased m6A level, restored p53 activation and inhibited cellular transformation phenotypes in the arsenite-transformed cells. Further, it was demonstrated that m6A downregulated the expression of the positive p53 regulator, PRDM2, through the YTHDF2-promoted decay of PRDM2 mRNAs. Further, m6A upregulated the expression of negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. 

Finally in 2021 a discovery that YTHDF2 is upregulated in NK cells upon activation by cytokines, tumors, and cytomegalovirus infection. Ythdf2 deficiency in NK cells impaired its anti-tumor and anti-viral activity in vivo. YTHDF2 maintains NK cell homeostasis and terminal maturation, correlating with modulating NK cell trafficking and regulating Eomes, respectively. YTHDF2 promotes NK cell effector function and is required for IL-15-mediated NK cell survival and proliferation by forming a STAT5-YTHDF2 positive feedback loop. Analysis showed significant enrichment in cell cycle, division, and division-related processes, including mitotic cytokinesis, chromosome segregation, spindle, nucleosome, midbody, and chromosome. This data supports roles of YTHDF2 in regulating NK proliferation, survival, and effector functions. Transcriptome-wide screening identified Tardbp (TDP-43) to be involved in cell proliferation or survival as a YTHDF2-binding target in NK cells.

Downregulation of METTL3, which in spinal cord contributes with YTHDF2 to modulate inflammatory pain may upregulate differentially expressed p53 network splice variants that oppose YTHDF2 induced downregulation of p53, via PRDM2 leading to apoptotic or diseased cells. In diseased environments cytokines may upregulate YTHDF2 in NK cells leading to downregulation of p53 and cytoskeletal transformation that may be sufficient, at an immune synapse to advance cytolysis.

p53 signals may inform selections of cells and tissue that prime NK cells for advanced, personalized immune therapy. 

Sunday, June 20, 2021

First Intron DNA - Site for a Genetic Brain?

DNA Methylation

The first intron of a gene, regardless of tissue or species is conserved as a site of downstream methylation with an inverse relationship to transcription and gene expression. Therefore, it is an informative gene feature regarding the relationship between DNA methylation and gene expression. But, expression in induced pluripotent stem cells (iPSC's) has been a major challenge to the stem cell industry, because by comparison these cells have not yet reached the state of natural pluripotent or embryonic stem cells (ESC's).

In mice two X chromosomes (XC) are active in the epiblasts of blastocysts as well as in pluripotent stem cells. One XC is inactivated triggered by Xist (non coding) RNA transcripts coating it to become silent. Designer transcription factor (dTF) repressors, binding the Xist intron 1 enhancer region caused higher H3K9me3 methylation and led to XC's opening and X-linked gene repression in MEFs. This substantially improved iPSC production and somatic cell nuclear transfer (SCNT) preimplantation embryonic development. This also correlated with much fewer abnormally expressed genes frequently associated with SCNT, even though it did not affect Xist expression. In stark contrast, the dTF activator targeting the same enhancer region drastically decreased both iPSC generation and SCNT efficiencies and induced ESC differentiation. 

A genome-wide, tissue-independent quasi-linear, inverse relationship exists between DNA methylation of the first intron and gene expression. More tissue-specific, differentially methylated regions exist in the first intron than in any other gene feature. These have positive or negative correlation with gene expression, indicative of distinct mechanisms of tissue-specific regulation. CpGs in transcription factor binding motifs are enriched in the first intron and methylation tends to increase with distance from the first exon–first intron boundary, with a concomitant decrease in gene expression.

Since the relationship between sequence, methylation, repression and transcription is determinative in ESC differentiation it may also suggest a broader link to differential translation. Translation is required for miRNA-dependent transcript destabilization that alters levels of coding and noncoding transcripts. But, steady-state abundance and decay rates of cytosolic long non-coding RNA's (lncRNAs) are insensitive to miRNA loss. Instead lncRNAs fused to protein-coding reporter sequences become susceptible to miRNA-mediated decay. 

In this model, first intron DNA sequences that are differentially methylated, bind transcription factors that effect transcription, impact splicing, expressions of coding or non-coding transcripts and transcript destabilizations resulting in differential rates and possible variations in translation. This bottom-up, dynamic view of the classical process may elevate the first intron from 'junk' to a DNA 'brain' because it plays a more extensive role, heading the process toward translation of any gene or switching it off entirely.  

For this reason, among others Codondex uses first intron k-mers relative to the transcripts mRNA as the basis for comparing same gene transcripts in diseased cells or tissue samples. Further, p53 and BRCA1 miRNA key sequences, discovered using Codondex iScore algorithm, when transfected into HeLa cells resulted in significantly reduced proliferation that may result from this accelerated, transfected miRNA dependent decay.

 

Tuesday, June 1, 2021

Short Sequences of Proximally Disordered DNA

Oxford Nanopore Device Reducing Sequencing Cost

Relationships exist between short sequences of proximal DNA (SSPD) of a gene that when transcribed into RNA present stronger or weaker binding attractions to RNA binding proteins (RBP'S) that settle, edit, splice and resolve messenger RNA (mRNA). Responsive to epigenetic stimuli on Histones and DNA, mRNA are constantly transcribed in different quantity, at different times such that different mRNA strands are transported from the nucleus to cytoplasm where they are translated into and produce any of more than 30,000 different proteins.

Single nucleotide polymorphisms and DNA mutations can alter SSPD combinations in different diseased cells thus altering sequence proximity, ordering that affects transcribed RNA's attraction and optimal binding of RBP's. This may result in modified splicing of RNA, assembly of mRNA and slight or major variations in some or all translated protein derived from that gene. 

The specific effects of these DNA variations, on the multitude of proteins produced are generally unknown. However, it remains important to understand their effects in disease, diagnosis and therapy. Typically these have historically been researched by large scale analysis of RBP on RNA as opposed to the more fundamental, yet underrepresented massive array of diseased variant DNA to mRNA transitions.

Most pharmaceutical research is directed to a molecular interference targeting an aberrant protein to cure widely represented or highly impactful disease conditions of society. Economic assessments generally influence government decisions to support research based on loss of GDP contribution by a specific disease in a  patient cohort. However, in the modern multi-omics era top down research into protein-RNA activity is descending deeper into the cell to include RNA-mRNA and mRNA-DNA customizable therapies that will eventually resolve individually assessed diseases at a price that addresses much larger array of patient needs.  

SNP's and other mutations can vary considerably in cells. These variations can cause instability during division and lead to translated differences that can ultimately drive cancerous cell growth to escape patient immunity. Like a 'whack-a-mole' game, pattern variation and mechanistic persistence eventually beat the player. Without effective immune clearance these cells can replicate into tumors and contribute to microenvironments that support their existence.

Link to video on tumor microenvironment https://youtu.be/Z9H2utcnBic

We thought to analyze DNA and mRNA transcripts from cells in tumors and their microenvironments to see if we could expose the SSPD disordered combinations that may have promoted sub-optimal RBP attractions and led to sustained immune escape. Given the complexity of DNA to mRNA transcription, for any given gene many distortions in gene data sets have to be filtered. To do that we focused on p53, the most mutated gene in cancer. We designed a method to compare sequences arrays of DNA and mRNA Ensembl transcripts, from the consensus of healthy patients to multiple cell samples extracted from different sections of a patients tumor and tumor microenvironment.     

We previously identified and measured different levels of Natural Killer (NK) cell cytotoxicity, produced from cocultures with the extracted samples of each of the multiple sites of a biopsy. We will measure the different p53 transcript SSPD combinations associated with each sample and determine whether disordered SSPD's corelate with NK cytotoxicity from each coculture. We expect to identify whether biopsied tumor cells, ranked by SSPD's predict the cytotoxicity resulting from NK cell cocultures. We will narrow our research to identify the varied expressions of receptor combinations associated with degrees of cytotoxicity. We will test immune efficacy to lyse and destroy tumor cells. Finally we will test for adaptive immune response. 

Our vision is for per-patient, predictable cell co-culture pairings, for innate immune cell education based on ranking DNA-mRNA combinations to lead to multiple effective therapies. The falling cost of sequencing and sophistication of GMP laboratories presently servicing oncologists may support a successful use of this analytical approach to laboratory assisted disease management.

   



 

Thursday, May 13, 2021

Non-Coding DNA Key Sequences

DNA Structural Inherency

Wind two strands of elastic, eventually it will knot, ultimately it will double up on itself. Separate the strands. From the point of unwinding forces will be directed to different regions and the separation will naturally return to the wound state of the band. Do the same with each of 10 different bands or strings of any type, they will all behave in much the same way. For a given section of DNA being transcribed, the effect of separation will be much the same. For a given gene, there will be sequences that can tolerate force to greater or lesser degrees. For different transcripts, of a gene variation at those sequences may be crucial to the integrity of transcription machinery that separates DNA strands to initiate replication to RNA and for the outcome.

Cellular biology is enormously complex in all regards. The physics of molecular interaction, fluid dynamics, and chemistry combine in a system where cause and effect is near impossible to predict. At the most elementary level we hypothesize some non-coding DNA (ncDNA) possess structural inherencies that can be deployed to direct gene proteins and cell function for diagnosis or therapy.

Coding DNA and its regulatory, non-coding gene compliment is transcribed and spliced from a transcribed gene. Transcription to RNA, edited mRNA, spliced non-coding RNA and ultimately mRNA translation to protein can produce wide ranging, variable outcomes that may not be re-captured experimentally. 

A single nucleotide polymorphism (SNP) or SNP combinations within a gene may affect the finely tuned balance that results. Under different environmental conditions this could be material to the protein produced. Additionally other mutations of the gene could add complexity to the environment and/or the  resulting protein translation. 

At this level of cellular biology, genetic DNA stores instruction for protein assemblies to produce new protein required for the fully functional cell. However, DNA's stored mutations can lead to different functional or non-functional versions of protein depending on many different factors. Relationships between ncDNA, including mutations and the transcripts' edited, protein coding mRNA may represent unexplored inherencies that can regulate the gene's mRNA or translated protein.

We built an algorithm to elaborately compare ncDNA sequences of multiple protein coding transcripts of the same gene. For each transcript it steps through every variable length ncDNA sequence (kmer) (specifically intron1), computes a signature for each and indexes it to the constant of the transcripts' mRNA signature. For each step these signatures order the kmers for each of the transcript's. The order is represented in a vector of all the transcripts being compared.  

At millions of successive steps (depending on total intron 1 length's) transcripts mostly retain their vector ordering except, as expected at a kmer length change. Mostly transcript order in the vector does not change, occasionally a few positions change, vary rarely do all positions change. Position changes that cause another, like a domino effect are filtered out. For the rarest positions changes at a step, we look to the root causes in the kmer (sequence). We call this a Key Sequence because it is identified by the significance of changes to transcript positions in the vector compared to the vector at the next step. 

Therefore, Key Sequences cause the most position changes between transcripts being compared by the algorithm. This relative measure is step dependent and Key Sequences are discovered by comparing transcript positions in the vector at the next step location. Logically, this infers a genes structural inherency discovered through ncDNA Key Sequence relationships to mRNA, to other transcripts, error in gene alignments, sequenced reads or the algorithm. 

In assay testing we were able to predict and synthesize non-coding RNA Key Sequences that significantly reduced proliferation of HeLa cells. In our pre-clinical work, based on comparisons to transcripts of the TP53 we will be predicting the efficacy of cell and tissue selections that educate and activate Natural Killer cells.

If Key Sequences are inherent they could open a new frontier for diagnosis and therapy.








Thursday, April 22, 2021

IFN-γ Concentration, p53 and Immune Sensitivity

IFN-γ 

Dimorphic complexity between Human Leukocyte Antigen (HLA) and Killer Immune Receptor (KIR) haplotypes
introduce significant challenges for personalized Natural Killer (NK) and immune cell therapy. In vitro models support a p53 requirement for upregulation of NK ligands and there is a strong association between the KIR B haplotype and p53 alteration in Basal Cell Carcinoma's (BCC) with a higher likelihood that KIR B carriers harbor abnormal p53. Data suggests that KIR encoded by B genes provides selective pressure for altered p53 in, at least BCC's. 

Breast cancer (BC) patients exhibit reduced NK-cytotoxicity in peripheral blood. To test whether certain KIR-HLA combinations impair NK-cytotoxicity that predispose to BC risk, KIR and HLA polymorphisms were analyzed in 162 women with BC and 278 controls. KIR-B genotypes increased significantly in BC. Certain activating KIR (aKIR) HLA ligand combinations were significantly increased in advanced-BC patients whose combinations also shared specific inhibitory KIR (iKIR) counterparts. Contrarily, iKIR-HLA pairs without their aKIR-HLA counterparts were significantly higher in controls. The data suggests NK cells expressing iKIR to cognate HLA-ligands in the absence of specific aKIR counterpart are instrumental in antitumor response. 

The TP53 family consists of three sets of transcription factor genes, TP53, TP63 and TP73, each expresses multiple RNA variants and protein isoforms. TP53 is mutated in 25-30% of BC's, but the effect of isoforms in BC is unknown. Predicted changes in expression of a subset of RNAs involved in IFN-γ signaling were confirmed in vitro. Data showed that different members of the TP53 family can drive transcription of genes involved in IFN-γ signaling in different BC subgroups. Moreover, tumors with low IFN-γ signaling were associated with significantly poorer patient outcome.

NK receptor NKG2D interacts with several virus or stress inducible ligands, including ULBP1 (NKG2DL1) and -2 expressed on target cells. Induction of wild-type p53, but not mutant p53, strongly upregulated mRNA and surface expression of ULBP1 and -2, but not other ligands. An intronic p53-responsive element was discovered in these genes. Coculture of wild-type, p53-induced human tumor cells with primary human NK cells enhanced NKG2D dependent degranulation and IFN-γ production by NK cells.  

In the Tumor Micro Environment (TME) IFN-γ is produced at various concentrations in response to numerous immune stimulants and highlights the need for more personalized, disease centric approach. Engagement of IFN-γ Receptor on distinct tumor stromal cells, induction of interferon stimulated genes, immune status of the TME, and IFN-γ concentration are recognized as critical determinants for IFN-γ-mediated outcomes. Notably, an appropriate antitumor concentration of IFN-γ has yet to be determined. Interestingly IFN-γ produced by NK cells is said to be an essential mediator of Angiotensin II inflammation and vascular dysfunction.

Pharmacological activation of p53 exerts a potent antileukemia effect on antitumor immunity, including NK cell-mediated cytotoxicity against acute myeloid leukemia (AML). Interestingly, orally administered DS-5272 (a potent inhibitor of MDM2 - promotor of p53 degradation) induced upregulation of CD107a and IFN-γ in NK cells but not in CD8+ T cells. Furthermore, coculture of NK cells with leukemia cells resulted in massive apoptosis. 

Findings strongly suggest an interaction between B7 (NK receptor) molecules contribute to a particular design of the inflammatory microenvironment including B7-H6 and PD-L1, for which therapy was enhanced by expanded NK autologous or donor cells. RNA transfections, into HeLa cells of p53 or BRCA1 intron1 Key Sequences (based on Codondex iScore's most significant mRNA-intron1 variations) caused several genes to be upregulated, +1500% above control including B7-H6 (NCR3LG1) ligand for NCR3 (Nkp30) NK cell receptor which, when engaged triggers IFN-γ release. NCR3 and soluble isoforms of Leukocyte Specific Transcript 1 may play a role in inflammatory and infectious diseases. 

Blockade of B7-H3 prolonged the survival of SKOV3 ovarian cancer cell, an in ovarian tumor-bearing mice, miR-29c improved the anti-tumor efficacy of NK-cell by directly targeting B7-H3. miR-29c downregulates B7-H3 and inhibits NK-cell exhaustion. Low levels of mir-29c have been associated with mutated p53 in BC patients. miR-29 miRNAs activate p53 by targeting p85α and CDC42 and upregulate p53 levels that induce apoptosis in a p53-dependent manner. miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting IFN-γ

Besides (intron predominant) human ALU repeats, reverse complementary sequences between introns bracketing circRNAs are highly enriched in RNA editing or hyper-editing events. Knockdown of double stranded RNA-editing enzyme - ADAR1 significantly and specifically upregulated circRNA expression. In its absence (interferon stimulating) oligoadenylate synthetase (OAS) can be activated by self-dsRNA (in contrast to viral dsRNA), resulting in RNase L activity and cell death. Conversely, OASL1 expression enhanced RIG-I-mediated IFN induction. In cells absent of p53, immunogenic, endogenous mitochondrial dsRNA are produced and processed by the OAS/RNase L system presenting a novel mechanism in diseases with aberrant immune responses. IFN-γ restores the impaired function of RNase L and induces mitochondria-mediated apoptosis in lung cancer. The p53—OAS axis, in mitochondrial RNA processing may prevent self-nucleic acid such as dsRNA from aberrantly activating innate immune responses.

A plethora of evidence supports bottom up approach to personalized therapy. A p53 intron1-mRNA regulatory loop, as a potential mechanism in IFN responses to infection and disease may be diagnostic. Pre-clinical research, presently underway will establish whether p53 is diagnostic for specific selections of a biopsy to educate NK cells and trigger effective immune response.