From single gene analysis to single cell profiling: a new era for precision medicine

Molecular profiling of DNA and RNA has provided valuable new insights into the genetic basis of non-malignant and malignant disorders, as well as an increased understanding of basic mechanisms that regulate human disease. Recent technological advances have enabled the analyses of alterations in gene-based structure or function in a comprehensive, high-throughput fashion showing that each tumor type typically exhibits distinct constellations of genetic alterations targeting one or more key cellular pathways that regulate cell growth and proliferation, evasion of the immune system, and other aspects of cancer behavior. These advances have important implications for future research and clinical practice in areas as molecular diagnostics, the implementation of gene or pathway-directed targeted therapy, and the use of such information to drive drug discovery. The 1st international and 32nd Annual Conference of Italian Association of Cell Cultures (AICC) conference wanted to offer the opportunity to match technological solutions and clinical needs in the era of precision medicine

The tumor suppressor gene KCTD11REN is regulated by Sp1 and methylation and its expression is reduced in tumors

A hallmark of several human cancers is loss of heterozygosity (LOH) of chromosome 17p13. The same chromosomal region is also frequently hypermethylated in cancer. Although loss of 17p13 has been often associated with p53 genetic alteration or Hypermethylated in Cancer 1 (HIC1) gene hypermethylation, other tumor suppressor genes (TSGs) located in this region have critical roles in tumorigenesis. A novel TSG mapping on human chromosome 17p13.2 is KCTD11REN (KCTD11). We have recently demonstrated that KCTD11 expression is frequently lost in human medulloblastoma (MB), in part by LOH and in part by uncharacterized epigenetic events. Using a panel of human 177 tumor samples and their normal matching samples representing 18 different types of cancer, we show here that the down-regulation of KCTD11 protein level is a specific and a diffusely common event in tumorigenesis. Additionally, in order to characterize the regulatory regions in KCTD11 promoter, we identified a CpG island and several Sp1 binding sites on this promoter, and demonstrated that Sp1 transcription factor and DNA methylation contribute, at least in part, to regulate KCTD11 expression. Our findings identify KCTD11 as a widely down-regulated gene in human cancers, and provide a basis to understand how its expression might be deregulated in tumor cells

Ren: a novel, developmentally regulated gene that promotes neural cell differentiation

Expansion and fate choice of pluripotent stem cells along the neuroectodermal lineage is regulated by a number of signals, including EGF, retinoic acid, and NGF, which also control the proliferation and differentiation of central nervous system (CNS) and peripheral nervous system (PNS) neural progenitor cells. We report here the identification of a novel gene, REN, upregulated by neurogenic signals (retinoic acid, EGF, and NGF) in pluripotent embryonal stem (ES) cells and neural progenitor cell lines in association with neurotypic differentiation. Consistent with a role in neural promotion, REN overexpression induced neuronal differentiation as well as growth arrest and p27Kip1 expression in CNS and PNS neural progenitor cell lines, and its inhibition impaired retinoic acid induction of neurogenin-1 and NeuroD expression. REN expression is developmentally regulated, initially detected in the neural fold epithelium of the mouse embryo during gastrulation, and subsequently throughout the ventral neural tube, the outer layer of the ventricular encephalic neuroepithelium and in neural crest derivatives including dorsal root ganglia. We propose that REN represents a novel component of the neurogenic signaling cascade induced by retinoic acid, EGF, and NGF, and is both a marker and a regulator of neuronal differentiation

The NF-κB pharmacopeia: novel strategies to subdue an intractable target

NF-κB transcription factors are major drivers of tumor initiation and progression. NF-κB signaling is constitutively activated by genetic alterations or environmental signals in many human cancers, where it contributes to almost all hallmarks of malignancy, including sustained proliferation, cell death resistance, tumor-promoting inflammation, metabolic reprogramming, tissue invasion, angiogenesis, and metastasis. As such, the NF-κB pathway is an attractive therapeutic target in a broad range of human cancers, as well as in numerous non-malignant diseases. Currently, however, there is no clinically useful NF-κB inhibitor to treat oncological patients, owing to the preclusive, on-target toxicities of systemic NF-κB blockade. In this review, we discuss the principal and most promising strategies being developed to circumvent the inherent limitations of conventional IκB kinase (IKK)/NF-κB-targeting drugs, focusing on new molecules that target upstream regulators or downstream effectors of oncogenic NF-κB signaling, as well as agents targeting individual NF-κB subunits

Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma

Copyright @ 2013 Macmillan Publishers Limited. This is the author's accepted manuscript. The final published article is available from the link below.Regulation of cell survival is a key part of the pathogenesis of multiple myeloma (MM). Jun N-terminal kinase (JNK) signaling has been implicated in MM pathogenesis, but its function is unclear. To elucidate the role of JNK in MM, we evaluated the specific functions of the two major JNK proteins, JNK1 and JNK2. We show here that JNK2 is constitutively activated in a panel of MM cell lines and primary tumors. Using loss-of-function studies, we demonstrate that JNK2 is required for the survival of myeloma cells and constitutively suppresses JNK1-mediated apoptosis by affecting expression of poly(ADP-ribose) polymerase (PARP)14, a key regulator of B-cell survival. Strikingly, we found that PARP14 is highly expressed in myeloma plasma cells and associated with disease progression and poor survival. Overexpression of PARP14 completely rescued myeloma cells from apoptosis induced by JNK2 knockdown, indicating that PARP14 is critically involved in JNK2-dependent survival. Mechanistically, PARP14 was found to promote the survival of myeloma cells by binding and inhibiting JNK1. Moreover, inhibition of PARP14 enhances the sensitization of MM cells to anti-myeloma agents. Our findings reveal a novel regulatory pathway in myeloma cells through which JNK2 signals cell survival via PARP14, and identify PARP14 as a potential therapeutic target in myeloma.Kay Kendall Leukemia Fund, NIH, Cancer Research UK, Italian Association for Cancer Research and the Foundation for Liver Research

Case report: Senescence as mechanism of resistance to Pembrolizumab in a Lymphoma patient who failed CD19-Targeted CAR-T cell therapy

BackgroundT cells engineered to target CD19 antigen on neoplastic B cells represent the most striking example of CAR-T cell therapy. The success rate of this therapy is affected by several limitations: target antigen loss, and/or acquisition of a senescent/exhausted phenotype by CAR and non-CAR T cells. Case presentationWe report on a patient affected by refractory Diffuse Large B-cell Lymphoma who was resistant to CAR T-cell therapy and to two cycles post CAR-T of pembrolizumab (PBZ) due to the evolution into a B-cell Hodgkin-like lymphoma. Owing to the CD30 expression and the Hodgkin-like phenotype, the patient was ultimately treated with Brentuximab-Vedotin and finally underwent remission. Upon PBZ treatment, 100% of circulating CAR-T+ cells showed a persistent CD8(+) senescent/exhausted phenotype, while an increase in the percentage of senescent cells was found in the non-CAR CD8(+) T cells compartment. ConclusionsPBZ is not able to reinvigorate exhausted CAR(+) T cells and to confer durable clinical response. We hypothesize that the phenomenon is due to the senescent phenotype of CAR(+) T cells, which did not allow PBZ-induced reactivation and proliferative rescue. The phenomenon, together with the loss of CAR-T target CD19 and the shift of non-CAR CD8(+) T cells towards a senescent phenotype likely contributed to set up an immune landscape with poor antitumor capacity

The glycosaminoglycan-binding domain of PRELP acts as a cell type–specific NF-κB inhibitor that impairs osteoclastogenesis

The PRELP heparin sulfate–binding protein translocates to the nucleus, where it impairs NF-κB transcriptional activity, which in turn regulates bone homeostasis

GADD45β loss ablates innate immunosuppression in cancer

T cell exclusion from the tumour microenvironment (TME) is a major barrier to overcoming immune escape. Here we identify a myeloid-intrinsic mechanism governed by the NF-κB effector molecule GADD45β that restricts tumour-associated inflammation and T cell trafficking into tumours. In various models of solid cancers refractory to immunotherapies, including hepatocellular carcinoma (HCC) and ovarian adenocarcinoma, Gadd45b inhibition in myeloid cells restored activation of pro-inflammatory tumour-associated macrophages (TAM) and intratumoural immune infiltration, thereby diminishing oncogenesis. Our results provide a basis to interpret clinical evidence that elevated expression of GADD45B confers poor clinical outcomes in most human cancers. Further, they suggest a therapeutic target in GADD45β for re-programming TAM to overcome immunosuppression and T cell exclusion from the TME