MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming.

Extensive desmoplasia and poor vasculature renders pancreatic tumors severely hypoxic, contributing to their aggressiveness and therapy resistance. Here, we identify the HuR/MYB/HIF1α axis as a critical regulator of the metabolic plasticity and hypoxic survival of pancreatic cancer cells. HuR undergoes nuclear-to-cytoplasmic translocation under hypoxia and stabilizes MYB transcripts, while MYB transcriptionally upregulates HIF1α. Upon MYB silencing, pancreatic cancer cells fail to survive and adapt metabolically under hypoxia, despite forced overexpression of HIF1α. MYB induces the transcription of several HIF1α-regulated glycolytic genes by directly binding to their promoters, thus enhancing the recruitment of HIF1α to hypoxia-responsive elements through its interaction with p300-dependent histone acetylation. MYB-depleted pancreatic cancer cells exhibit a dramatic reduction in tumorigenic ability, glucose-uptake and metabolism in orthotopic mouse model, even after HIF1α restoration. Together, our findings reveal an essential role of MYB in metabolic reprogramming that supports pancreatic cancer cell survival under hypoxia.

Biphasic transcriptional and posttranscriptional regulation of MYB by androgen signaling mediates its growth control in prostate cancer.

MYB, a proto-oncogene, is overexpressed in prostate cancer (PCa) and promotes its growth, aggressiveness, and resistance to androgen-deprivation therapy. Here, we examined the effect of androgen signaling on MYB expression and delineated the underlying molecular mechanisms. Paralleling a dichotomous effect on growth, low-dose androgen induced MYB expression at both transcript and protein levels, whereas it was suppressed in high-dose androgen-treated PCa cells. Interestingly, treatment with both low- and high-dose androgen transcriptionally upregulated MYB by increasing the binding of androgen receptor to the MYB promoter. In a time-course assay, androgen induced MYB expression at early time points followed by a sharp decline in high-dose androgen-treated cells due to decreased stability of MYB mRNA. Additionally, profiling of MYB-targeted miRNAs demonstrated significant induction of miR-150 in high-dose androgen-treated PCa cells. We observed a differential binding of androgen receptor on miR-150 promoter with significantly greater occupancy recorded in high-dose androgen-treated cells than those treated with low-dose androgen. Functional inhibition of miR-150 relieved MYB suppression by high-dose androgen, while miR-150 mimic abolished MYB induction by low-dose androgen. Furthermore, MYB-silencing or miR-150 mimic transfection suppressed PCa cell growth induced by low-dose androgen, whereas miR-150 inhibition rescued PCa cells from growth repression by high-dose androgen. Similarly, we observed that MYB silencing suppressed the expression of androgen-responsive, cell cycle-related genes in low-dose androgen-treated cells, while miR-150 inhibition increased their expression in cells treated with high-dose androgen. Overall, these findings reveal novel androgen-mediated mechanisms of MYB regulation that support its biphasic growth control in PCa cells.

Modulation of the tumor microenvironment by natural agents: implications for cancer prevention and therapy.

The development of cancer is not just the growth and proliferation of a single transformed cell, but its surrounding environment also coevolves with it. Indeed, successful cancer progression depends on the ability of the tumor cells to develop a supportive tumor microenvironment consisting of various types of stromal cells. The interactions between the tumor and stromal cells are bidirectional and mediated through a variety of growth factors, cytokines, metabolites, and other biomolecules secreted by these cells. Tumor-stromal crosstalk creates optimal conditions for the tumor growth, metastasis, evasion of immune surveillance, and therapy resistance, and its targeting is being explored for clinical management of cancer. Natural agents from plants and marine life have been at the forefront of traditional medicine. Numerous epidemiological studies have reported the health benefits imparted on the consumption of certain fruits, vegetables, and their derived products. Indeed, a significant majority of anti-cancer drugs in clinical use are either naturally occurring compounds or their derivatives. In this review, we describe fundamental cellular and non-cellular components of the tumor microenvironment and discuss the significance of natural compounds in their targeting. Existing literature provides hope that novel prevention and therapeutic approaches will emerge from ongoing scientific efforts leading to the reduced tumor burden and improve clinical outcomes in cancer patients.

Single-cell RNA sequencing reveals peripheral blood mononuclear immune cell landscape associated with operational tolerance in a kidney transplant recipient.

Operational tolerance (OT) after kidney transplantation is defined as stable graft acceptance without the need for immunosuppression therapy. However, it is not clear which cellular and molecular pathways are driving tolerance in these patients. In this first-of-its-kind pilot study, we assessed the immune landscape associated with OT using single-cell analyses. Peripheral mononuclear cells from a kidney transplant recipient with OT (Tol), 2 healthy individuals (HC), and a kidney transplant recipient with normal kidney function on standard-of-care immunosuppression (SOC) were evaluated. The immune landscape of the Tol was drastically different from that of SOC and emerged closer to the profile of HC. TCL1A+ naive B cells and LSGAL1+ regulatory T cells (Tregs) were in higher proportions in Tol. We were unable to identify the Treg subcluster in SOC. The ligand-receptor analysis in HC and Tol identified interactions between B cells, and Tregs that enhance the proliferation and suppressive function of Tregs. SOC reported the highest proportion of activated B cells with more cells in the G2M phase. Our single-cell RNA sequencing study identified the mediators of tolerance; however, it emphasizes the requirement of similar investigations on a larger cohort to reaffirm the role of immune cells in tolerance.

European Society for Organ Transplantation Consensus Statement on Biomarkers in Liver Transplantation.

Currently, one-year survival following liver transplantation (LT) exceeds 90% in large international registries, and LT is considered definitive treatment for patients with end-stage liver disease and liver cancer. Recurrence of disease, including hepatocellular carcinoma (HCC), significantly hampers post-LT outcomes. An optimal approach to immunosuppression (IS), including safe weaning, may benefit patients by mitigating the effect on recurrent diseases, as well as reducing adverse events associated with over-/under-IS, including chronic kidney disease (CKD). Prediction of these outcome measures-disease recurrence, CKD, and immune status-has long been based on relatively inaccurate clinical models. To address the utility of new biomarkers in predicting these outcomes in the post-LT setting, the European Society of Organ Transplantation (ESOT) and International Liver Transplant Society (ILTS) convened a working group of experts to review literature pertaining to primary disease recurrence, development of CKD, and safe weaning of IS. Summaries of evidence were presented to the group of panelists and juries to develop guidelines, which were discussed and voted in-person at the Consensus Conference in Prague November 2022. The consensus findings and recommendations of the Liver Working Group on new biomarkers in LT, clinical applicability, and future needs are presented in this article.

MYB interacts with androgen receptor, sustains its ligand-independent activation and promotes castration resistance in prostate cancer.

BACKGROUND: Aberrant activation of androgen receptor signalling following castration therapy is a common clinical observation in prostate cancer (PCa). Earlier, we demonstrated the role of MYB overexpression in androgen-depletion resistance and PCa aggressiveness. Here, we investigated MYB-androgen receptor (AR) crosstalk and its functional significance. METHODS: Interaction and co-localization of MYB and AR were examined by co-immunoprecipitation and immunofluorescence analyses, respectively. Protein levels were measured by immunoblot analysis and enzyme-linked immunosorbent assay. The role of MYB in ligand-independent AR transcriptional activity and combinatorial gene regulation was studied by promoter-reporter and chromatin immunoprecipitation assays. The functional significance of MYB in castration resistance was determined using an orthotopic mouse model. RESULTS: MYB and AR interact and co-localize in the PCa cells. MYB-overexpressing PCa cells retain AR in the nucleus even when cultured under androgen-deprived conditions. AR transcriptional activity is also sustained in MYB-overexpressing cells in the absence of androgens. MYB binds and promotes AR occupancy to the KLK3 promoter. MYB-overexpressing PCa cells exhibit greater tumorigenicity when implanted orthotopically and quickly regain growth following castration leading to shorter mice survival, compared to those carrying low-MYB-expressing prostate tumours. CONCLUSIONS: Our findings reveal a novel MYB-AR crosstalk in PCa and establish its role in castration resistance.

Co-targeting of CXCR4 and hedgehog pathways disrupts tumor-stromal crosstalk and improves chemotherapeutic efficacy in pancreatic cancer.

Pancreatic cancer (PC) remains a therapeutic challenge because of its intrinsic and extrinsic chemoresistance mechanisms. Here, we report that C-X-C motif chemokine receptor 4 (CXCR4) and hedgehog pathways cooperate in PC chemoresistance via bidirectional tumor-stromal crosstalk. We show that when PC cells are co-cultured with pancreatic stellate cells (PSCs) they are significantly more resistant to gemcitabine toxicity than those grown in monoculture. We also demonstrate that this co-culture-induced chemoresistance is abrogated by inhibition of the CXCR4 and hedgehog pathways. Similarly, the co-culture-induced altered expression of genes in PC cells associated with gemcitabine metabolism, antioxidant defense, and cancer stemness is also reversed upon CXCR4 and hedgehog inhibition. We have confirmed the functional impact of these genetic alterations by measuring gemcitabine metabolites, reactive oxygen species production, and sphere formation in vehicle- or gemcitabine-treated monocultures and co-cultured PC cells. Treatment of orthotopic pancreatic tumor-bearing mice with gemcitabine alone or in combination with a CXCR4 antagonist (AMD3100) or hedgehog inhibitor (GDC-0449) displays reduced tumor growth. Notably, we show that the triple combination treatment is the most effective, resulting in nearly complete suppression of tumor growth. Immunohistochemical analysis of Ki67 and cleaved caspase-3 confirm these findings from in vivo imaging and tumor measurements. Our findings provide preclinical and mechanistic evidence that a combination of gemcitabine treatment with targeted inhibition of both the CXCR4 and hedgehog pathways improves outcomes in a PC mouse model.

Proteomic Analysis of MYB-Regulated Secretome Identifies Functional Pathways and Biomarkers: Potential Pathobiological and Clinical Implications.

Earlier we have shown important roles of MYB in pancreatic tumor pathobiology. To better understand the role of MYB in the tumor microenvironment and identify MYB-associated secreted biomarker proteins, we conducted mass spectrometry analysis of the secretome from MYB-modulated and control pancreatic cancer cell lines. We also performed in silico analyses to determine MYB-associated biofunctions, gene networks, and altered biological pathways. Our data demonstrated significant modulation (p < 0.05) of 337 secreted proteins in MYB-silenced MiaPaCa cells, whereas 282 proteins were differentially present in MYB-overexpressing BxPC3 cells, compared to their respective control cells. Alteration of several phenotypes such as cellular movement, cell death and survival, inflammatory response, protein synthesis, etc. was associated with MYB-induced differentially expressed proteins (DEPs) in secretomes. DEPs from MYB-silenced MiaPaCa PC cells were suggestive of the downregulation of genes primarily associated with glucose metabolism, PI3K/AKT signaling, and oxidative stress response, among others. DEPs from MYB-overexpressing BxPC3 cells suggested the enhanced release of proteins associated with glucose metabolism and cellular motility. We also observed that MYB positively regulated the expression of four proteins with potential biomarker properties, i.e., FLNB, ENO1, ITGB1, and INHBA. Mining of publicly available databases using Oncomine and UALCAN demonstrated that these genes are overexpressed in pancreatic tumors and associated with reduced patient survival. Altogether, these data provide novel avenues for future investigations on diverse biological functions of MYB, specifically in the tumor microenvironment, and could also be exploited for biomarker development.

Hypoxia alters the release and size distribution of extracellular vesicles in pancreatic cancer cells to support their adaptive survival.

Pancreatic tumors are highly desmoplastic and poorly-vascularized, and therefore must develop adaptive mechanisms to sustain their survival under hypoxic condition. Extracellular vesicles (EV) play vital roles in pancreatic tumor pathobiology by facilitating intercellular communication. Here we studied the effect of hypoxia on the release of EVs and examined their role in adaptive survival of pancreatic cancer (PC) cells. Hypoxia promoted the release of EV in PC cell lines, MiaPaCa and AsPC1, wherein former exhibited a far greater induction. Moreover, a time-dependent, measurable and significant increase was recorded for small EV (SEV) in both the cell lines with only minimal induction observed for medium (MEV) and large EVs (LEV). Similarly, noticeable changes in size distribution of SEV were also recorded with a shift toward smaller average size under extreme hypoxia. Thrombospondin (apoptotic bodies marker) was exclusively detected on LEVs, while Arf6 (microvesicles marker) was mostly present on MEV with some expression in LEV as well. However, CD9 and CD63 (exosome markers) were expressed in both SEV and MEVs with a decreased expression recorded under hypoxia. Among all subfractions, SEV was the most bioactive in promoting the survival of hypoxic PC cells and hypoxia-inducible factor-1α stabilization was involved in heightened EV release under hypoxia and for their potency to promote hypoxic cell survival. Altogether, our findings provide a novel mechanism for the adaptive hypoxic survival of PC cells and should serve as the basis for future investigations on broader functional implications of EV.

Epigenetic basis of cancer health disparities: Looking beyond genetic differences.

Despite efforts at various levels, racial health disparities still exist in cancer patients. These inequalities in incidence and/or clinical outcome can only be explained by a multitude of factors, with genetic basis being one of them. Several investigations have provided convincing evidence to support epigenetic regulation of cancer-associated genes, which results in the differential transcriptome and proteome, and may be linked to a pre-disposition of individuals of certain race/ethnicity to early or more aggressive cancers. Recent technological advancements and the ability to quickly analyze whole genome have aided in these efforts, and owing to their relatively easy detection, methylation events are much well-characterized, than the acetylation events, across human populations. The early trend of investigating a pre-determined set of genes for differential epigenetic regulation is paving way for more unbiased screening. This review summarizes our current understanding of the epigenetic events that have been tied to the racial differences in cancer incidence and mortality. A better understanding of the epigenetics of racial diversity holds promise for the design and execution of novel strategies targeting the human epigenome for reducing the disparity gaps.

Exosomes confer chemoresistance to pancreatic cancer cells by promoting ROS detoxification and miR-155-mediated suppression of key gemcitabine-metabolising enzyme, DCK.

BACKGROUND: Chemoresistance is a significant clinical problem in pancreatic cancer (PC) and underlying molecular mechanisms still remain to be completely understood. Here we report a novel exosome-mediated mechanism of drug-induced acquired chemoresistance in PC cells. METHODS: Differential ultracentrifugation was performed to isolate extracellular vesicles (EVs) based on their size from vehicle- or gemcitabine-treated PC cells. Extracellular vesicles size and subtypes were determined by dynamic light scattering and marker profiling, respectively. Gene expression was examined by qRT-PCR and/or immunoblot analyses, and direct targeting of DCK by miR-155 was confirmed by dual-luciferase 3'-UTR reporter assay. Flow cytometry was performed to examine the apoptosis indices and reactive oxygen species (ROS) levels in PC cells using specific dyes. Cell viability was determined using the WST-1 assay. RESULTS: Conditioned media (CM) from gemcitabine-treated PC cells (Gem-CM) provided significant chemoprotection to subsequent gemcitabine toxicity and most of the chemoresistance conferred by Gem-CM resulted from its EVs fraction. Sub-fractionation grouped EVs into distinct subtypes based on size distribution and marker profiles, and exosome (Gem-Exo) was the only sub-fraction that imparted chemoresistance. Gene expression analyses demonstrated upregulation of SOD2 and CAT (ROS-detoxifying genes), and downregulation of DCK (gemcitabine-metabolising gene) in Gem-Exo-treated cells. SOD/CAT upregulation resulted, at least in part, from exosome-mediated transfer of their transcripts and they suppressed basal and gemcitabine-induced ROS production, and partly promoted chemoresistance. DCK downregulation occurred through exosome-delivered miR-155 and either the functional suppression of miR-155 or restoration of DCK led to marked abrogation of Gem-Exo-mediated chemoresistance. CONCLUSIONS: Together, these findings establish a novel role of exosomes in mediating the acquired chemoresistance of PC.

Hydroxytyrosol Induces Apoptosis and Cell Cycle Arrest and Suppresses Multiple Oncogenic Signaling Pathways in Prostate Cancer Cells.

SCOPE: Hydroxytyrosol (HT), a polyphenol from olives, is a potential anticancer agent. This study was designed to evaluate the anticancer activity of HT against prostate cancer cells, and the mechanism thereof. METHODS AND RESULTS: Treatment of LNCaP and C4-2 prostate cancer cells with HT resulted in a dose-dependent inhibition of proliferation. This was in contrast to HT's ineffectiveness against normal prostate epithelial cells RWPE1 and PWLE2, suggesting cancer-cell-specific effect. HT induced G1/S cell cycle arrest, with inhibition of cyclins D1/E and cdk2/4 and induction of inhibitory p21/p27. HT also induced apoptosis, as confirmed by flow cytometry, caspase activation, PARP cleavage, and BAX/Bcl-2 ratio. It inhibited the phosphorylation of Akt/STAT3, and induced cytoplasmic retention of NF-κB, which may explain its observed effects. Finally, HT inhibited androgen receptor (AR) expression and the secretion of AR-responsive prostate-specific antigen. CONCLUSION: Castration-resistant prostate cancers retain AR signaling and are often marked by activated Akt, NF-κB, and STAT3 signaling. Our results establish a pleiotropic activity of HT against these oncogenic signaling pathways. Combined with its nontoxic effects against normal cells, our results support further testing of HT for prostate cancer therapy.

Molecular Drivers of Pancreatic Cancer Pathogenesis: Looking Inward to Move Forward.

Pancreatic cancer (PC) continues to rank among the most lethal cancers. The consistent increase in incidence and mortality has made it the seventh leading cause of cancer-associated deaths globally and the third in the United States. The biggest challenge in combating PC is our insufficient understanding of the molecular mechanism(s) underlying its complex biology. Studies during the last several years have helped identify several putative factors and events, both genetic and epigenetic, as well as some deregulated signaling pathways, with implications in PC onset and progression. In this review article, we make an effort to summarize our current understanding of molecular and cellular events involved in the pathogenesis of pancreatic malignancy. Specifically, we provide up-to-date information on the genetic and epigenetic changes that occur during the initiation and progression of PC and their functional involvement in the pathogenic processes. We also discuss the impact of the tumor microenvironment on the molecular landscape of PC and its role in aggressive disease progression. It is envisioned that a better understanding of these molecular factors and the mechanisms of their actions can help unravel novel diagnostic and prognostic biomarkers and can also be exploited for future targeted therapies.

Cancer Chemoprevention by Phytochemicals: Nature's Healing Touch.

Phytochemicals are an important part of traditional medicine and have been investigated in detail for possible inclusion in modern medicine as well. These compounds often serve as the backbone for the synthesis of novel therapeutic agents. For many years, phytochemicals have demonstrated encouraging activity against various human cancer models in pre-clinical assays. Here, we discuss select phytochemicals-curcumin, epigallocatechin-3-gallate (EGCG), resveratrol, plumbagin and honokiol-in the context of their reported effects on the processes of inflammation and oxidative stress, which play a key role in tumorigenesis. We also discuss the emerging evidence on modulation of tumor microenvironment by these phytochemicals which can possibly define their cancer-specific action. Finally, we provide recent updates on how low bioavailability, a major concern with phytochemicals, is being circumvented and the general efficacy being improved, by synthesis of novel chemical analogs and nanoformulations.

Simulating hypoxia-induced acidic environment in cancer cells facilitates mobilization and redox-cycling of genomic copper by daidzein leading to pro-oxidant cell death: implications for the sensitization of resistant hypoxic cancer cells to therapeutic challenges.

This study was conducted to investigate the mechanism of action involved in the anti-cancer activity of daidzein and identification of cancer specific micro-environment as therapeutic target of this secondary metabolite derived from soy. Our data indicated that daidzein induces cellular DNA breakage, anti-proliferative effects and apoptosis in a concentration-dependent manner. We demonstrated that such a daidzein-induced anti-cancer action involves a copper-dependant pathway in which endogenous copper is mobilized by daidzein and redox-cycled to generate reactive oxygen species which act as an upstream signal leading to pro-oxidant cell death. Further in the context of hypoxia being a resistant factor against standard therapies and that an effect secondary to hypoxia is the intracellular acidification, we show that the anticancer activity of daidzein is modulated positively in acidic pH but copper-specific chelator is still able to inhibit daidzein activity. Moreover, an experimental setup of hypoxia mimic (cobalt chloride) revealed an enhanced sensitivity of cancer cells to the cytotoxic effects of daidzein which was neutralized in the presence of neocuproine. The findings support a paradigm shift from the conventional antioxidant property of dietary isoflavones to molecules capable of initiating a pro-oxidant signaling mediated by reactive oxygen species. Further, the clinical relevance of such an action mechanism in cancer chemoprevention is also proposed. This study identified endogenous copper as a molecular target and acidic pH as a modulating factor for the therapeutic activity of daidzein against cancer. The evidence presented highlights the potential of dietary agents as adjuvants to standard therapeutic regimens.

Mobilization of Intracellular Copper by Gossypol and Apogossypolone Leads to Reactive Oxygen Species-Mediated Cell Death: Putative Anticancer Mechanism.

There is compelling evidence that serum, tissue and intracellular levels of copper are elevated in all types of cancer. Copper has been suggested as an important co-factor for angiogenesis. It is also a major metal ion present inside the nucleus, bound to DNA bases, particularly guanine. We have earlier proposed that the interaction of phenolic-antioxidants with intracellular copper leads to the generation of reactive oxygen species (ROS) that ultimately serve as DNA cleaving agents. To further validate our hypothesis we show here that the antioxidant gossypol and its semi-synthetic derivative apogossypolone induce copper-mediated apoptosis in breast MDA-MB-231, prostate PC3 and pancreatic BxPC-3 cancer cells, through the generation of ROS. MCF10A breast epithelial cells refractory to the cytotoxic property of these compounds become sensitized to treatment against gossypol, as well as apogossypolone, when pre-incubated with copper. Our present results confirm our earlier findings and strengthen our hypothesis that plant-derived antioxidants mobilize intracellular copper instigating ROS-mediated cellular DNA breakage. As cancer cells exist under significant oxidative stress, this increase in ROS-stress to cytotoxic levels could be a successful anticancer approach.

Redox cycling of Cu(II) by 6-mercaptopurine leads to ROS generation and DNA breakage: possible mechanism of anticancer activity.

6-Mercaptopurine (6MP) is a well-known purine antimetabolite used to treat childhood acute lymphoblastic leukemia and other diseases. Cancer cells as compared to normal cells are under increased oxidative stress and show high copper level. These differences between cancer cells and normal cells can be targeted to develop effective cancer therapy. Pro-oxidant property of 6MP in the presence of metal ions is not well documented. Redox cycling of Cu(II) to Cu(I) was found to be efficiently mediated by 6MP. We have performed a series of in vitro experiments to demonstrate the pro-oxidant property of 6MP in the presence of Cu(II). Studies on human lymphocytes confirmed the DNA damaging ability of 6MP in the presence of Cu(II). Since 6MP possesses DNA damaging ability by producing reactive oxygen species (ROS) in the presence of Cu(II), it may also possess apoptosis-inducing activity by involving endogenous copper ions. Essentially, this would be an alternative and copper-dependent pathway for anticancer activity of 6MP.

Insights into the Role of microRNAs in Pancreatic Cancer Pathogenesis: Potential for Diagnosis, Prognosis, and Therapy.

Pancreatic cancer is a highly lethal malignancy and a fourth leading cause of cancer-related death in the United States. Poor survival of pancreatic cancer patients is largely because of its asymptomatic progression to advanced stage against which no effective therapy is currently available. Over the years, we have developed significant knowledge of molecular progression of pancreatic cancer and identified several genetic and epigenetic aberrations to be involved in its etiology and aggressive behavior. In that regard, recent lines of evidence have suggested important roles of microRNAs (miRNAs/miRs) in pancreatic cancer pathogenesis. microRNAs belonging to a family of small, noncoding RNAs are able to control diverse biological processes due to their ability to regulate gene expression at the posttranscriptional level. Accordingly, dysregulation of miRNAs can lead to several disease conditions, including cancer. There is a long list of microRNAs that exhibit aberrant expression in pancreatic cancer and serve as key microplayers in its initiation, progression, metastasis, and chemoresistance. These findings have suggested that microRNAs could be exploited as novel biomarkers for diagnostic and prognostic assessments of pancreatic cancer and as targets for therapy. This book chapter describes clinical problems associated with pancreatic cancer, roles that microRNAs play in various aspects of pancreatic cancer pathogenesis, and envision opportunities for potential use of microRNAs in pancreatic cancer management.

Contribution of Proteomics in Transplantation: Identification of Injury and Rejection Markers.

Solid organ transplantation saves thousands of lives suffering from end-stage diseases. Although early transplants experienced acute organ injury, medical breakthroughs, such as tissue typing, and use of immunosuppressive agents have considerably improved graft survival. However, the overall incidence of allograft injury and chronic rejection remains high. Often the clinical manifestations of organ injury or rejection are nonspecific and late. Current requirement for successful organ transplantation is the identification of reliable, accurate, disease-specific, noninvasive methods for the early diagnosis of graft injury or rejection. Development of noninvasive techniques is important to allow routine follow-ups without the discomfort and risks associated with a graft biopsy. Multiple biofluids have been successfully tested for the presence of potential proteomic biomarkers; these include serum, plasma, urine, and whole blood. Kidney transplant research has provided significant evidence to the potential of proteomics-based biomarkers for acute and chronic kidney rejection, delayed graft function, early detection of declining allograft health. Multiple proteins have been implicated as biomarkers; however, recent observations implicate the use of similar canonical pathways and biofunctions associated with graft injury/rejection with altered proteins as potential biomarkers. Unfortunately, the current biomarker studies lack high sensitivity and specificity, adding to the complexity of their utility in the clinical space. In this review, we first describe the high-throughput proteomics technologies and then discuss the outcomes of proteomics profiling studies in the transplantation of several organs. Existing literature provides hope that novel biomarkers will emerge from ongoing efforts and guide physicians in delivering specific therapies to prolong graft survival.

Dynamics of Age- versus Therapy-Related Clonal Hematopoiesis in Long-term Survivors of Pediatric Cancer.

We present the first comprehensive investigation of clonal hematopoiesis (CH) in 2,860 long-term survivors of pediatric cancer with a median follow-up time of 23.5 years. Deep sequencing over 39 CH-related genes reveals mutations in 15% of the survivors, significantly higher than the 8.5% in 324 community controls. CH in survivors is associated with exposures to alkylating agents, radiation, and bleomycin. Therapy-related CH shows significant enrichment in STAT3, characterized as a CH gene specific to survivors of Hodgkin lymphoma, and TP53. Single-cell profiling of peripheral blood samples revealed STAT3 mutations predominantly present in T cells and contributed by SBS25, a mutational signature associated with procarbazine exposure. Serial sample tracking reveals that larger clone size is a predictor for future expansion of age-related CH clones, whereas therapy-related CH remains stable decades after treatment. These data depict the distinct dynamics of these CH subtypes and support the need for longitudinal monitoring to determine the potential contribution to late effects. SIGNIFICANCE: This first comprehensive CH analysis in long-term survivors of pediatric cancer presents the elevated prevalence and therapy exposures/diagnostic spectrum associated with CH. Due to the contrasting dynamics of clonal expansion for age-related versus therapy-related CH, longitudinal monitoring is recommended to ascertain the long-term effects of therapy-induced CH in pediatric cancer survivors. See related commentary by Collord and Behjati, p. 811. This article is highlighted in the In This Issue feature, p. 799.