In Vivo Syngeneic Tumor Models with Acquired Resistance to Anti-PD-1/PD-L1 Therapies.

Antibodies targeting PD-1 and PD-L1 have produced durable responses in a subset of patients with cancer. However, a majority of these patients will ultimately relapse due to acquired resistance. To explore the underlying mechanisms of this secondary resistance, we developed five syngeneic murine tumor variants with acquired resistance to anti-PD-1 and/or PD-L1 antibodies in vivo. Resistant in vivo models were obtained by serial treatment/reimplantation cycles of the MC38 colorectal, MB49 and MBT2 bladder, and RENCA kidney and TyrNras melanoma models. Tumor immune infiltrates were characterized for wild type and resistant tumors using spectral cytometry and their molecular alterations analyzed using RNA sequencing analyses. Alterations in the tumor immune microenvironment were strongly heterogeneous among resistant models, involving select lymphoid and/or myeloid subpopulations. Molecular alterations in resistant models included previously identified pathways as well as novel candidate genes found to be deregulated in several resistant models. Among these, Serpinf1, coding for pigment epithelial-derived factor (PEDF) was further explored in the MC38 and the MBT2 models. Overexpression of Serpinf1 induced resistance to anti-PD-1 antibodies in the MC38 model, whereas knockdown of Serpinf1 sensitized this model as well as the primarily resistant MBT2 model. Serpinf1 overexpression was associated with increased production of free fatty acids and reduced activation of CD8+ cells, while orlistat, a compound that reduces the production of free fatty acids, reversed resistance to anti-PD-1 therapy. Our results suggest that a panel of syngeneic resistant models constitutes a useful tool to model the heterogeneity of resistance mechanisms encountered in the clinic.

MAVS deficiency induces gut dysbiotic microbiota conferring a proallergic phenotype.

Prominent changes in the gut microbiota (referred to as "dysbiosis") play a key role in the development of allergic disorders, but the underlying mechanisms remain unknown. Study of the delayed-type hypersensitivity (DTH) response in mice contributed to our knowledge of the pathophysiology of human allergic contact dermatitis. Here we report a negative regulatory role of the RIG-I-like receptor adaptor mitochondrial antiviral signaling (MAVS) on DTH by modulating gut bacterial ecology. Cohousing and fecal transplantation experiments revealed that the dysbiotic microbiota of Mavs-/- mice conferred a proallergic phenotype that is communicable to wild-type mice. DTH sensitization coincided with increased intestinal permeability and bacterial translocation within lymphoid organs that enhanced DTH severity. Collectively, we unveiled an unexpected impact of RIG-I-like signaling on the gut microbiota with consequences on allergic skin disease outcome. Primarily, these data indicate that manipulating the gut microbiota may help in the development of therapeutic strategies for the treatment of human allergic skin pathologies.

The thyroid hormone nuclear receptor TRα1 controls the Notch signaling pathway and cell fate in murine intestine.

Thyroid hormones control various aspects of gut development and homeostasis. The best-known example is in gastrointestinal tract remodeling during amphibian metamorphosis. It is well documented that these hormones act via the TR nuclear receptors, which are hormone-modulated transcription factors. Several studies have shown that thyroid hormones regulate the expression of several genes in the Notch signaling pathway, indicating a possible means by which they participate in the control of gut physiology. However, the mechanisms and biological significance of this control have remained unexplored. Using multiple in vivo and in vitro approaches, we show that thyroid hormones positively regulate Notch activity through the TRα1 receptor. From a molecular point of view, TRα1 indirectly controls Notch1, Dll1, Dll4 and Hes1 expression but acts as a direct transcriptional regulator of the Jag1 gene by binding to a responsive element in the Jag1 promoter. Our findings show that the TRα1 nuclear receptor plays a key role in intestinal crypt progenitor/stem cell biology by controlling the Notch pathway and hence the balance between cell proliferation and cell differentiation.

Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance.

The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1's function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells.

Thyroid hormones and their nuclear receptors: new players in intestinal epithelium stem cell biology?

Thyroid hormones participate in the development and homeostasis of several organs and tissues. It is well documented that they act via nuclear receptors, the TRs, which are transcription factors whose function is modulated by the hormone T3. Importantly, T3-induced physiological response within a cell depends on the specific TR expression and on the T3 bioavailability. However, in addition to this T3-dependent control of TR functionality, increasing data show that the action of TRs is coordinated and integrated with other signaling pathways, specifically at the level of stem/progenitor cell populations. By focusing on the intestinal epithelium of both amphibians and mammals we summarize here new data in support of a role for thyroid hormones and the TR nuclear receptors in stem cell biology. This new concept may be extended to other organs and have biological relevance in therapeutic approaches aimed to target stem cells such as tissue engineering and cancer.

The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A.

Coordination of cell cycle events in space and time is crucial to achieve a successful cell division. Here, we demonstrate that UBXN-2, a substrate adaptor of the AAA ATPase Cdc48/p97, is required to coordinate centrosome maturation timing with mitosis. In UBXN-2-depleted Caenorhabditis elegans embryos, centrosomes recruited more AIR-1 (Aurora A), matured precociously, and alignment of the mitotic spindle with the axis of polarity was impaired. UBXN-2 and CDC-48 coimmunoprecipitated with AIR-1 and the spindle alignment defect was partially rescued by co-depleting AIR-1, indicating that UBXN-2 controls these processes via AIR-1. Similarly, depletion in human cells of the UBXN-2 orthologues p37/p47 resulted in an accumulation of Aurora A at centrosomes and a delay in centrosome separation. The latter defect was also rescued by inhibiting Aurora A. We therefore postulate that the role of this adaptor in cell cycle regulation is conserved.

Aurora A in cell division: kinase activity not required.

Aurora A kinase is a key regulator of cell division, whose functions were attributed to its ability to phosphorylate diverse substrates. Aurora A is now shown to have a kinase-independent role in the regulation of chromatin-mediated microtubule assembly.

Cooperation between the thyroid hormone receptor TRalpha1 and the WNT pathway in the induction of intestinal tumorigenesis.

BACKGROUND & AIMS: Colorectal tumorigenesis is a multistep process involving the alteration of oncogenes and tumor suppressor genes, leading to the deregulation of molecular pathways that govern intestinal homeostasis. We have previously shown that the thyroid hormone receptor alpha1 (TRalpha1) controls intestinal development and homeostasis through the WNT pathway. More precisely, TRalpha1 directly enhances the transcription of several components of this pathway, allowing increased expression of beta-catenin/Tcf4 target genes and stimulation of cell proliferation. Because the WNT pathway is a major player in controlling intestinal homeostasis, we addressed whether the TRalpha1 receptor has tumor-inducing potential. METHODS: We generated mice overexpressing TRalpha1 specifically in the intestinal epithelium in a wild-type (vil-TRalpha1) or a WNT-activated (vil-TRalpha1/Apc(+/1638N)) genetic background. RESULTS: The intestine of vil-TRalpha1 mice presents aberrant intestinal mucosal architecture and increased cell proliferation and develops adenoma at a low rate. However, TRalpha1 overexpression is unable to induce cancer development. On the contrary, we observed accelerated tumorigenesis in vil-TRalpha1/Apc(+/1638N) mice compared with the Apc(+/1638N) mutants. CONCLUSION: Our results suggest that this phenotype is due to cooperation between the activated TRalpha1 and WNT pathways. This is the first report describing the tumor-inducing function of TRalpha1 in the intestine.

Thyroid hormones and the control of cell proliferation or cell differentiation: paradox or duality?

Amphibian metamorphosis perfectly illustrates a key paradox: thyroid hormones control diverse cellular processes depending on the tissue context. This point is also reinforced by a recent accumulation of evidence. For example, thyroid hormones and their nuclear receptor TRs have been described to function in different systems in synergy and/or in antagonism with other signaling pathways. This interaction helps explain their pleiotropic roles. This review summarizes the most important advances in this field, focusing in particular on the key action of thyroid hormones in controlling the balance between the processes of cell proliferation and cell differentiation in a few organs, with special attention paid to the intestine. We highlight similarities between the cellular and molecular events occurring during postnatal intestinal maturation at metamorphosis in amphibians, and comparable events observed at weaning in mice.

The frizzled-related sFRP2 gene is a target of thyroid hormone receptor alpha1 and activates beta-catenin signaling in mouse intestine.

The thyroid hormone receptor TRalpha1 regulates intestinal development and homeostasis by controlling epithelial proliferation in the crypts. This involves positive control of the Wnt/beta-catenin pathway. To further investigate the effect of thyroid hormone-TRalpha1 signaling on the intestinal epithelium proliferating compartment, we performed a comparative transcription profile analysis on laser microdissected crypt cells recovered from wild type animals with normal or perturbed hormonal status, as well as from TR knock-out mice. Statistical analysis and an in silico approach allowed us to identify 179 differentially regulated genes and to group them into organized functional networks. We focused on the "cell cycle/cell proliferation" network and, in particular, on the Frizzled-related protein sFRP2, whose expression was greatly increased in response to thyroid hormones. In vitro and in vivo analyses showed that the expression of sFRP2 is directly regulated by TRalpha1 and that it activates beta-catenin signaling via Frizzled receptors. Indeed, sFRP2 stabilizes beta-catenin, activates its target genes, and enhances cell proliferation. In conclusion, these new data, in conjunction with our previous results, indicate a complex interplay between TRalpha1 and components of the Wnt/beta-catenin pathway. Moreover, we describe in this study a novel mechanism of action of sFRP2, responsible for the activation of beta-catenin signaling.

The thyroid hormone receptor-alpha (TRalpha) gene encoding TRalpha1 controls deoxyribonucleic acid damage-induced tissue repair.

The thyroid hormone (TH) controls, via its nuclear receptor, TH receptor-alpha1 (TRalpha1), intestinal crypt cell proliferation in the mouse. In order to understand whether this receptor also plays a role in intestinal regeneration after DNA damage, we applied a protocol of gamma-ray irradiation and monitored cell proliferation and apoptosis at several time points. In wild-type mice, the dose of 8 Gy induced cell cycle arrest and apoptosis in intestinal crypts a few hours after irradiation. This phenomenon reverted 48 h after irradiation. TRalpha(0/0) mutant mice displayed a constant low level of proliferating cells and a high apoptosis rate during the period of study. At the molecular level, in TRalpha(0/0) animals we observed a delay in the p53 phosphorylation induced by DNA damage. In our search for the expression of the protein kinases responsible for p53 phosphorylation upon irradiation, we have focused on DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The number of cells expressing DNA-PKcs in crypts remained high 48 h after irradiation, specifically in TRalpha mutants. Altogether, in TRalpha(0/0) animals the rate of apoptosis in crypt cells remained high, apparently due to an elevated number of cells still presenting DNA damage. In conclusion, the TRalpha gene plays a role in crypt cell homeostasis by regulating the rate of cell renewal and apoptosis induced by DNA damage.

Thyroid hormone receptor alpha1 directly controls transcription of the beta-catenin gene in intestinal epithelial cells.

Thyroid hormones, T3 and T4, are known regulators of intestine development. The best characterized example is the remodeling of the gastrointestinal tract during amphibian metamorphosis. Thyroid hormones act via nuclear receptors, the TRs, which are T3-dependent transcription factors. We previously showed that intestinal epithelial cell proliferation is controlled by thyroid hormones and the TRalpha gene. To analyze the mechanisms responsible, we studied the expression of genes belonging to and/or activated by the Wnt/beta-catenin pathway, a major actor in the control of physiological and pathological epithelial proliferation in the intestine. We show that T3-TRalpha1 controls the transcription of the beta-catenin gene in an epithelial cell-autonomous way. This is parallel to positive regulation of proliferation-controlling genes such as type D cyclins and c-myc, known targets of the Wnt/beta-catenin. In addition, we show that the regulation of the beta-catenin gene is direct, as TR binds in vitro and in chromatin in vivo to a specific thyroid hormone-responsive element present in intron 1 of this gene. This is the first report concerning in vivo transcriptional control of the beta-catenin gene. As Wnt/beta-catenin plays a crucial role in intestinal tumorigenesis, our observations open a new perspective on the study of TRs as potential tumor inducers.

Critical role of hnRNP A1 in HTLV-1 replication in human transformed T lymphocytes.

BACKGROUND: In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1. RESULTS: We have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown in C91PL cells increased viral production by these cells. Thus, hnRNP A1 is implicated in the modulation of the level of HTLV-1 gene expression in T cells transformed by this human retrovirus. CONCLUSIONS: These observations provide an insight into a new cellular control of HTLV-1 replication and suggest that hnRNP A1 is likely part of the regulatory mechanisms of the life cycle of this human retrovirus in T cells.